Artificial immune system: methods for making and use

ABSTRACT

The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the normal tissues that interact with vaccines in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interaction with the immune system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit as a divisional of U.S. application Ser.No. 11/116,234, filed Apr. 28, 2005, which claims priority from U.S.Provisional Application Ser. No. 60/565,846, filed Apr. 28, 2004 andU.S. Provisional Application Ser. No. 60/643,175 filed Jan. 13, 2005.The entirety of each of these applications is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

A portion of the work described herein was supported by contract numberDAMD 17-02-C-0130 and contract number DARPA #BAA03-02 from theDepartment of Defense. The United States Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a method for constructing anintegrated artificial human tissue and, in particular, construction ofan integrated human immune system for in vitro testing of vaccines,adjuvants, immunotherapy candidates, cosmetics, drugs, biologics, andother chemicals. The artificial immune system is useful for assessingthe interaction of substances with the immune system, and thus can beused to accelerate and improve the accuracy of vaccine, drug, biologic,immunotherapy, cosmetic, and chemical development.

2. Background of the Technology

The development and biological testing of human vaccines hastraditionally relied on small animal models, such as mouse and rabbitmodels, and then non-human primate models. However, such small animalmodels are expensive and non-human primate models are both expensive andprecious.

The mammalian immune system uses two general adaptive mechanisms toprotect the body against environmental pathogens. When apathogen-derived molecule is encountered, the immune response is highlyactivated to ensure protection against that pathogenic organism.

The first mechanism is the non-specific (or innate) inflammatoryresponse. The innate immune system appears to recognize specificmolecules that are present on pathogens but not on the body itself.

The second mechanism is the specific or acquired (or adaptive) immuneresponse. Innate responses are fundamentally the same for each injury orinfection. In contrast, acquired responses are custom tailored to thepathogen in question. The acquired immune system evolves a specificimmunoglobulin (antibody) response to many different molecules presentin the pathogen, called antigens. In addition, a large repertoire of Tcell receptors is sampled for their ability to bind processed forms ofthe antigens bound to MHC class I and II on antigen-presenting cells(APCs), such as dendritic cells (DCs).

The immune system recognizes and responds to structural differencesbetween self and non-self proteins. Proteins that the immune systemrecognizes as non-self are referred to as antigens. Pathogens typicallyexpress large numbers of highly complex antigens. Acquired immunity hasspecific memory for antigenic structures; repeated exposure to the sameantigen increases the response, which increases the level of inducedprotection against that particular pathogen.

Acquired immunity is mediated by specialized immune cells called B and Tlymphocytes (or simply B and T cells). B cells produce and mediate theirfunctions through the actions of antibodies. B cell-dependent immuneresponses are referred to as “humoral immunity,” because antibodies aredetected in body fluids. T cell-dependent immune responses are referredto as “cell mediated immunity,” because effector activities are mediateddirectly by the local actions of effector T cells. The local actions ofeffector T cells are amplified through synergistic interactions betweenT cells and secondary effector cells, such as activated macrophages. Theresult is that the pathogen is killed and prevented from causingdiseases.

Similar to pathogens, vaccines function by initiating an innate immuneresponse at the vaccination site and activating antigen-specific T and Bcells that can give rise to long term memory cells in secondary lymphoidtissues. The precise interactions of the vaccine with cells at thevaccination site and with T and B cells of the lymphoid tissues areimportant to the ultimate success of the vaccine.

Almost all vaccines to infectious organisms were and continue to bedeveloped through the classical approach of generating an attenuated orinactivated pathogen as the vaccine itself. This approach, however,fails to take advantage of the recent explosion in our mechanisticunderstanding of immunity. Rather, it remains an empirical approach thatconsists of making variants of the pathogen and testing them forefficacy in non-human animal models.

Advances in the design, creation and testing of more sophisticatedvaccines have been stalled for several reasons. First, only a smallnumber of vaccines can be tested in humans, because, understandably,there is little tolerance for harmful side effects in healthy childrenexposed to experimental vaccines. With the exception of cancer vaccinetrials, this greatly limits the innovation that can be allowed in thereal world of human clinical trials. Second, it remains challenging topredict which epitopes are optimal for induction of immunodominant CD4and CD8 T cell responses and neutralizing B cell responses. Third, smallanimal testing, followed by primate trials, has been the mainstay ofvaccine development; such approaches are limited by intrinsicdifferences between human and non-human species, and ethical and costconsiderations that restrict the use of non-human primates.Consequently, there is a slow translation of basic knowledge to theclinic, but equally important, a slow advance in the understanding ofhuman immunity in vivo.

The artificial immune system (AIS) of the present invention can be usedto address this inability to test many novel vaccines in human trials byinstead using human tissues and cells. The AIS enables rapid vaccineassessment in an in vitro model of human immunity. The AIS provides anadditional model for testing vaccines in addition to the currently usedanimal models.

Attempts have been made in modulating immune system. See, for example,U.S. Pat. No. 6,835,550 B1; U.S. Pat. No. 5,008,116; Suematsu et al.,Nat. Biotechnol., 22, 1539-1545 (2004); and US Patent Publication No.2003/0109042.

Nevertheless, none of these publications describes or suggests the AIS,which comprises a vaccine site (VS), a lymphoid tissue equivalent (LTE),a lymphatic and blood vascular network equivalent, and the use of AISfor assessing the interaction of substances with the immune system.

SUMMARY OF THE INVENTION

The present invention provides an integrated system of the functionallyequivalent human tissues for testing vaccines, adjuvants, drugs,biologics, cosmetics, and other chemicals in vitro. One aspect of theinvention relates to a method for constructing a functionally equivalenttissue using blueprints that design, as opposed to fabricate,morphologically equivalent constructs. Functional equivalency to thehuman immune system is achieved by building three engineered tissueconstructs (ETCs), housed in a modular miniaturized immunobioreactorsystem.

Another aspect of the invention relates to a method of constructing anartificial immune system. The method comprises: (1) designing andblueprinting three functionally equivalent immunologic engineeredtissues that form the basis for the human immune system (vaccinationsite, lymphoid tissue equivalent, and lymphatic/vascular highways); (2)providing real time communication pathways between the engineeredimmunological constructs; and (3) integrating the engineered tissues andimmunological constructs in a modular immunobioreactor to form the basisfor an in vitro AIS for rapid vaccine assessment.

Approaches to construction of the artificial immune system include theconstruction of engineered immunological tissues, populated with areproducible cell source, with a particular focus on dendritic cells(DCs). The ability to optimize the spatial juxtaposition and temporalrelationships between the cells, biomolecules, and scaffolds via adirected self assembly process moves far beyond existing two dimensional(2D) Petri dish cell cultures into reproducible three dimensional, (3D)heterogeneous, biologically viable constructs, much more similar to thein vivo situation.

The artificial immune system of the present invention further relates tothe method of using AIS for (1) modulating the immune system in asubject to eliminate various of infectious diseases and pathogens (2)rapid comparison of vaccine or immunotherapy formulations; (3) rationaldissection of vaccine or immunotherapy action to identify “rate limitingsteps” to focus further development; and (4) systematic determination ofoptimal formulations to create better vaccines that promote more rapidand long lived protection.

The predictive value of such an engineered tissue construct equivalentimmune system is superior to current in vitro models of human immunity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) is a schematic representation of the LTE in which T and B cellare cultivated together on microcarriers and then transferred to aporous container

FIG. 1(B) is a schematic representation of the LTE in which T and B cellare cultivated on separate microcarriers and then brought together in aporous container.

FIG. 1(C) is a schematic representation of the LTE in which separate Tand B cell microcarriers are cultivated on separate microcarriers andthen brought together in a porous container with separate compartments.

FIG. 1(D FIG. 1D is a schematic showing binding T and B cells to themicrocarriers having CXC 13 ligand as an adhesion ligand.

FIG. 1(E) illustrates the influence of microcarrier particle size on theporosity and openings between the microcarriers.

FIG. 2 illustrates how the AIS of the present invention operates.

FIG. 3 is a schematic representation of vaccination site (VS).

FIG. 4 is a schematic representation of an artificial lymphoid tissue(or lymphoid tissue equivalent).

FIG. 5. A schematic of the in vitro immune system ensemble.

FIG. 6A are example micrographs of microspheres encapsulatingfluorochrome-labeled fMLP chemokine (a monocyte and immature DCchemoattractant).

FIG. 6B are diagrams showing in vitro release kinetics for a peptidechemokine (fMLP) and a 10 kDa chemokine (MIP-3a).

FIGS. 7A-7D demonstrate in vitro control of monocyte and dendritic cellchemotaxis. FIG. 7A is a schematic representation of the experimentaldesign. FIG. 7B shows the movement of monocyte-derived DCs in theabsence of chemokine. FIGS. 7C and 7D show the movement ofmonocyte-derived DCs and moncytes, respectively, towards the vaccinationsite source well.

FIGS. 8A and 8B are pictures showing cell development in a lymphoidtissue equivalent (LTE) matrix. Synthetic inverse opal hydrogelscaffolds were synthesized that support T-cell migration and interactionwith B cells (FIG. 8A), and the attachment and growth of high celldensities, which is an important feature in mimicking themicroenvironment of the lymph node (FIG. 8B).

FIG. 9 shows mockup of digitally printed lymph node (left panel) and aretinal image of vasculature (right panel).

FIGS. 10A-10C show antigen-presenting cell behavior in an artificialimmune system. FIG. 10A shows pictures of vascular endothelial cellsgrown on 3D constructs of fibronectin-coated collagen. FIG. 10B is aschematic diagram showing the stages of monocyte behavior in such a 3Dculture. FIG. 10C shows demonstrates that monocytes can be infected withinfluenza to measure activation of IFNγ induction and expansion duringrecall responses in T cells from adults previously infected with flu.

FIG. 11 is a schematic representing a 3D model of a vaccination site(VS).

FIG. 12 is a picture of a porous scaffold prepared using the method ofExample 6.

FIG. 13 is a picture of a porous scaffold prepared using the method ofExample 7.

FIG. 14 is a picture of a porous scaffold prepared using the method ofExample 8.

FIG. 15 show pictures of confluent HUVEC culture on the bottom (left)and top (right) surfaces of a porous Protasan/collagen membranesupported by a nylon mesh.

FIG. 16 illustrates monocytes migrated through the two-side HUVECculture grown on porous Protasan/collagen mesh-supported membrane. Left:Monocytes on the bottom of the chamber without MCP-1, underneath of thetwo-side HUVEC culture, 30 min after application onto the membrane.Right: Monocytes on the bottom of the chamber with MCP-1, 30 min afterapplication onto the membrane.

FIG. 17 is a picture showing HUVEC culture growing on a bovine collagentype I membrane.

FIG. 18 is a composite of pictures showing human monocytes permeated theHUVEC culture on a collagen mesh-supported matrix.

FIG. 19 is a picture of synthetic and natural membranes supported bystainless steel rings.

FIG. 20 is a schematic representation of an embodiment of an in vitrovaccination site.

FIG. 21 is a schematic picture of a rapid chemokine testing system.

FIGS. 22A and 22B show the structure of a lymph node. FIG. 22A is aschematic view of a lymph node. FIG. 22B is a histologic section of alymph node; B cells are stained blue and T cells are stained brown(Abbas, et al., Cellular and Molecular Immunology (W.B. Saunders Co.,New York, N.Y.) (2000)).

FIGS. 23A-23I are phase contrast micrographs of a confluent monolayer ofHUVEC cells on a collagen cushion. FIG. 23A is an image of toluidineblue-stained HUVEC cells on a collagen cushion. FIG. 23B is a highermagnification of FIG. 23A. FIG. 23C shows a high density of newlyapplied peripheral blood mononuclear cells (PBMCs) on the layer ofHUVEC. FIG. 23D shows a focal plane below the HUVEC cells, within thecollagen matrix, 45 minutes after the application of PBMCs. Cells infocus are within the collagen and are easily distinguished between HUVECand surface PBMCs. FIG. 23E is an image of CMFDA labeling showing cellviability and position of live cells within the collagen cushion. FIG.23F shows transmigration of PBMCs into collagen cushions without or withthe presence of Zymosan. Phase contrast, and CMFDA labeling was done todetermine cell placement within the cushion. Z-stack images were takenthrough the entire cushion to determine the numbers of cells within thecushion and those that had undergone transmigration. FIG. 23G showsincreased numbers of transmigrated cells remained in the cushion in thepresence of Zymosan as compared to cushions with no Zymosan. FIG. 23H isa comparison of depth of penetration in the presence of Zymosan versusno Zymosan. FIG. 231 is a schematic diagram of this experimental design.

FIG. 24A-24C show organization of the T-zone of the lymph node. FIG. 24Ashows a schematic of overall lymph node structure and a scanningelectron micrograph of ECM structure in the T zone (Gretz, et al.,Immunol. Rev. 156:11-24 (1997)). FIG. 24B is a confocalimmunofluorescence image of stromal cells lining the reticular network(Gretz, et al., J. Exp. Med. 192:1425-1440 (2000)). Collagen fibers ofthe network are stained red, while reticular fibroblasts are stainedgreen. FIG. 24C shows fiber organization in an in vitro type I collagengel (Kaldjian, et al., Int. Immunol. 13:1243-1253 (2001); Friedl, etal., Eur. J. Immunol., 28:2331-2343 (1998)).

FIG. 25 shows a model of B cell activation in lymph nodes (fromBaumgarth Immunol. Rev. 176:171-180, (2000)).

FIG. 26 shows a potential HEV model for the in vitro model immunesystem. A separate monolayer in the plane of the HEV surface would becomposed of lymphatic endothelium to provide to separate access pointsto the LTE.

FIGS. 27A-27C show matrix design for the lymphoid tissue equivalent. (A)approach for fabricating ordered scaffolds for the LTE. (B) Examplebright field micrograph showing the highly ordered nature of thehydrogel scaffold (view through several layers observed in situ inmedium). (C) Chemistry for surface bio functionalization of matrices.Shown on the right is a fluorescence micrograph of a scaffoldfunctionalized with FITC labeled fibronectin.

FIG. 28 shows image of microbeads fabricated from lymphoid ECM (80% w/w)and Protasan (20% w/w) by flash freezing, freeze drying, and gelationwith tripolyphosphate.

FIG. 29(A) shows image of B cells on Cytodex-1 microcarriers (Amersham),bright field microscopy (left) and fluorescence (right).

FIG. 29(B) shows image of T cells on Cytopore-1 microcarriers(Amersham), bright field (left) and fluorescence (right).

FIG. 29(C) shows confocal fluorescence image of T cells on LNECM/Protasan in-house microcarriers.

FIG. 30 shows the structure of heparin, a natural component ofextracellular matrix. Heparin contains multiple pentasaccharide unitsbearing sulfate and carboxylic groups; the average charge per unit ratiois 2.3.

FIG. 31 illustrates that heparin-treated Cytopore possesses amplesorption capacity for the BLC chemokine (FIG. 31A), and steeptime-release curve (FIG. 31B). Thus, it is a suitable carrier for Band/or T cells.

FIG. 32 demonstrates fibroblastic reticular cell line derivation. (A)phase contrast micrograph of a confluent monolayer of T zone FRCs inculture. (B) flow cytometry measurement of CD44 and VCAM-1 expression byFRCs. (C) micrograph of FRC growth on flat RGD-PEG hydrogel surfaces.

FIG. 33 illustrates a mock lymph node. (A) direct printing of aheterogeneous matrix. Example of a digitally-printed mock ‘lymph node’structure. (B) schematic of digital printing assembly of heterogeneousLTE with T- and B-zones, using co-deposited controlled releasemicrospheres to maintain lymphocyte localization.

FIG. 34 shows a lymphoid tissue equivalent (LTE). (A) maintenance of Tcell and B cell areas in vivo. (B) schematic of T cell-DC localizationto T cell areas of the LTE via MIP-3β-releasing microspheres.

FIG. 35 shows an additional embodiment involving ‘templating’ the LTEusing native human stromal cells in a manner similar to that reported byresearchers attempting to create an in vitro artificial thymus(Poznansky, et al., Nat. Biotechnol. 18:729-734, (2000)).

FIG. 36 is a schematic of a bioreactor.

FIG. 37 shows a laminate based insert whereas a larger milled tubulardesign is incorporated in to the design illustrated in FIG. 36.

FIG. 38 shows an example microfluidic bioreactor with opticaldiagnostics on microfluidic backplane.

FIG. 39 shows an embodiment of the MaAIS.

FIG. 40 shows laser machined integrated optical waveguides: n1represents the refractive index of the waveguide core, n₂ is thecladding index.

FIG. 41 shows images of cells captured using Dynabeads M450. Left:T-lymphocyte (from Safarik & Safarikova, Rev. J. Chromatog, (1999) B,722:33-53; DYNAL (Norway); Right: MCF-7 breast cancer cell (from Sieben,et al.).

FIG. 42 is a schematic of a “Magnetic broom” used to move cells from VSto LTE.

FIG. 43 is a schematic showing magnetic bead assisted ELISA ofantibodies in the LTE compartment.

FIG. 44 is a schematic showing hypothetical hyper conjugate of the antiCD3 magnetic bead with CD4+ and CD8+ T cells and fluorescent anti-CD4and anti-CD8 antibodies.

FIG. 45 shows the role of CCR8 in DC migration: role for CCR8immigration. Panels A and C are graphs showing migration of monocytes inthe absence or presence anti-CCR8 mAb 3B10. Panels B and D show monocyteconversion into DCs in vivo using green fluorescent latex microspheresinjected into the skin of CCR8-deficient and age/sex-matched wild-typeC57BL/6 counterparts.

FIG. 46 shows images of an ultra-short pulse laser micromachined planaroptical waveguides integrated into microfluidic channel. Left panel:Tapered port for fiber optic coupling. Middle panel: microfluidicchannel intersection of planar waveguide (source off). Right panel:microfluidic channel intersection of planar waveguide (source on,entering from right).

FIG. 47 is a composite of pictures showing seeding of endothelial cellson both sides of an amniotic membrane. Panels A and B areimmunohistological staining of respective endothelial monolayers.Blue=endothelial nuclei; Green=CD31 staining to identify confluentendothelial junctions; Red=acting to identify actin bundles in all celltypes and particularly fibroblasts. Some fibroblasts are visible beneaththe vascular endothelium. Panel C is immunohistological staining offibroblasts within the amnion. Panel D is immunohistological staining ofmonocytes that have traversed the blood endothelium and moved toward thesecond endothelial monolayer. Panel E is hematoxylin staining of anendothelial monolayer. Panels F and G show that monocytes penetratedeeply into the amniotic membrane toward the second endothelialmonolayer.

FIG. 48 is a plan view of an example integrated bioreactor that showsmicromachined blood vascular and lymphatic pathways with high contactarea (left panel) beneath the VS and LTE ETCs (right panel).

FIG. 49 shows cross sectional views of direct deposition in the AISdevice. Various biomaterial structures can be incorporated asconstituents of the artificial immune system (e.g., bio concrete,inverse hydrogel opal, colloidal particles, ECM gels, collagen gels,microcarriers). For example, a polymeric mesh rebar can be depositedlayer by layer directly in the recessions of the VS and LTE areas. Insuch a design, it is preferred to have the lower plate of the AIS unitmade of polyacrylate, polystyrene, or another transparent plasticsensitive to DM, to allow the mesh rebar to attach to the plate. In thisembodiment, the surface is micro-patterned using KOH in a manner similarto the ESC scaffolds. Fibrin gel matrix bearing all necessary nutrientsand cytokines can be used to coat the threads of the mesh as a thinfilm, leaving sufficient space for cell accommodation and motion.

FIG. 50 shows an example microfluidic bioreactor in separate layers.

FIG. 51 shows an assembled microfluidic bioreactor.

FIG. 52 is a schematic diagram of perfused bioreactor system with theassociated external pumps for the lymphatic and blood vascular loops andexternal media reservoirs. The AIS bioreactor can be operated in eithersemi-batch or continuous mode.

FIG. 53 shows membranes between thin metal (e.g., stainless steel)rings. Using such a crimping method, biological membranes can besupported without use of adhesives and can be pressed into a disk withthickness profile of about 400 μm or less.

FIG. 54 is a schematic showing the fabrication of a 3-layer planarwaveguide.

FIG. 55 shows an example device comprising a perfusion bioreactor, anELISA chip with integrated optical waveguides, microfluidic backplane toconnect and allow swapping of devices and microfluidic connectors forexternal pumps and reservoirs.

FIG. 56 shows phagocytosis of microparticles by a monocyte.

FIG. 57 (A) and (B) show kinetics in silico modeling of T and Binteraction in LTE. FIG. 57 (A) is the in silico 2D models of B and Tcell interaction. Size of cell is 12 μm; size of carrier is 250 μm;speed of T cell is 12 μm/min; speed of B cell: 6 μm/min; B-T interactiontime is 3 min; cell population density: 2.76×10⁷/ml (1/40 space taken bycells); number of B cells are 65; and number of T cells are 65.

FIG. 58 shows characterization of the HUVEC endothelial cells in thecollagen cushion. Panels A-C: HUVEC cells before (A) and after (B; C)staining with CMFDA to determine cell viability by epifluorescentillumination prior to application of PBMCs for migration studies. PanelD: characterization of HUVEC cells by phase contrast. Panels E and F,staining with CMFDA showing live cells (E) and with ethidium homodimer-1showing dead cells (F).

FIG. 59 shows an example bioreactor construction with collagen membraneson rings and support matrix. Panel A sows the bioreactor design. Panel Bshows progression from the whole bioreactor to the level of the collagenmatrix cushion within the mesh. Panel C shows the assembly of thebioreactor under sterile conditions, after the HUVEC cells have reachedconfluence on the collagen cushion. Once assembled, media flow isinitiated.

FIG. 60 shows the preparation of an inverse opal gel scaffold. Panel A:Inverse opal gel scaffolds can be prepared with arbitrary dimensions bythe choice of mold for creating the colloidal crystal template. Shown isa photograph of a templated gel ˜6 mm in diameter and ˜2 mm tall, on theend of a spatula (coin for scale). Panel B: Gels of very precisethicknesses can be fabricated by templating gels, polymerizingadditional gel material around the templated scaffold, and then slicingthe bulk-gel-surrounded construct into arbitrary thin layers.

FIG. 61 shows T cell motility induction on inverse opal scaffolds. PanelA: 3D reconstruction of naive T cells (green) migrating over clusters ofmature dendritic cells (red) within voids of the inverse opal scaffold.Times in the upper corners represent relative elapsed min:sec.Color-coded arrows track the position of several cells in the field ofview. Panel B: Deconvolved 2D fluorescence image demonstrating the rapidtrafficking of one naive T cells (green, marked with arrow) from onecluster of DCs to another (clusters identified with dotted lines infirst frame), laterally through a window connecting two voids of thescaffold. Elapsed time as in panel (A).

FIG. 62 shows quantitation of cell movement. Panel A: Single-cellinstantaneous velocities of cells migrating in scaffolds vs. time. PanelB: 2D projections of single cell migration paths in x and y, withpositions shown in microns, plotted over 30 min T cells show randommigration within the scaffold, as observed in the lymph node T zone.Panel C: Mean displacement as a function of time for naive T cells ininverse opal scaffolds, co-cultured with mature dendritic cells.

FIG. 63 illustrate that Cell-cell contact alone within scaffolds doesnot drive T cell migration. Naive OT-II CD4+ T cells cultured at lymphnode-like cell densities in inverse opal scaffolds do not polarize ormigrate. Panel A: overall view of a region of scaffold by bright fieldmicroscopy. Panel B: 3 time-lapse clips of cells within one void of thematrix. Times are elapsed min:sec.

FIG. 64 shows HUVEC cells growing on protasan/collagen matrix on a nylonmesh. High-magnification SEM of the nylon membrane and interspersedProtasan/collagen matrix material is shown in the top image. Seeding ofthe primary layer of HUVEC cells was accomplished on an invertedmembrane (left, Side 1), then 24 hours later, brought to an uprightposition (right, Side 2) where the second layer was applied. Phasecontrast images of each plane of HUVEC cells is shown in the center twolower images, with the left being the first layer, and the right beingthe second layer applied.

FIG. 65 show pictures of ring structures showing variable methods ofattachment of membranes for VS in the bioreactor. The left panel showsthe spiked ring design used to hold ‘wet’ membrane structures such asamniotic or UBM naturally occurring ECM membranes. The right panel showsthree methods used to attach ‘dry’ synthetic membranes to the ringstructure. Top left (next to the left side of the dime) is crimped,bottom left is by laminating the membrane between two rings of the samematerial, and bottom right (below the dime) is glued.

FIG. 66 shows HUVEC cells on the culture plate with a bead of Devontwo-part epoxy applied and polymerized in place prior to seeding.

FIG. 67 is a sorption curve of Cytopore/heparin.

DETAILED DESCRIPTION OF THE INVENTION

A primary objective of the present invention is to provide an integratedhuman tissue, an integrated human immune system, for testing vaccines,immunotherapies, adjuvants, drugs, biologics, cosmetics, and otherchemicals in vitro. One aspect of the invention relates to methods toconstruct an integrated human immune system model that comprise usingappropriate in vitro cellular and tissue constructs or their equivalentsto mimic the normal tissues that interact with vaccines in humans. Suchan integrated platform of human tissues enables acceleration of vaccinedevelopment strategies and testing of drugs that interact with theimmune system. Furthermore, it enables a reduction in animal testing andenables candidate vaccines to be re-engineered and retested at afraction of the cost of animal studies and human trials.

Tissue engineering involves the development of synthetic materials ordevices that are capable of specific interactions with cells andtissues. The constructs combine these materials with living cells toyield functional tissue equivalents. Tissue engineering involves anumber of different disciplines, such as biomaterial engineering, drugdelivery, recombinant DNA techniques, biodegradable polymers,bioreactors, stem cell isolation, cell encapsulation and immobilization,and the production of 2D and 3D scaffolds for cells. Porousbiodegradable biomaterial scaffolds are required for the 3D growth ofcells to form the tissue engineering constructs. There are severaltechniques to obtain porosity for the scaffolds, including fiberbonding, solvent casting/particulate leaching, gas foaming/particulateleaching, and liquid-liquid phase separation. These produce large,interconnected pores to facilitate cell seeding and migration. As usedherein, the terms “tissue-engineered construct” or “engineered tissueconstruct” (“ETC”) include any combination of naturally derived orsynthetically grown tissue or cells, along with a natural or syntheticscaffold that provides structural integrity to the construct.

It is known that 3D biology is important to induce proper functionalityof immunological ETCs (see, e.g., Edelman & Keefer, Exp. Neurol. 192:1-6(2005). A principal approach to studying cellular processes is toculture cells in vitro. Historically, this has involved plating cells onplastic or glass supports. Cells grown on solid or filter support arereferred as two dimensional (2D) cultures. Such 2D cultures on poroussupports have been extremely useful for studying many aspects ofbiology. However, much more in vivo-like conditions can now be realizedin 3D cultures. For example, many epithelial cells, both primarycultures and established lines, form complex epithelial structures whengrown in 3D ECM.

Recently, in lymph nodes, it has been shown that 3D interstitial tissuematrix facilitates not only T cell migration toward an APC, but alsosupports motility upon cell-cell interaction. A 3D collagen matrixenvironment, because of its spatial architecture, provides traction forlymphocyte crawling, mimicking some structural features of the lymphnode cortex. This provides experimental justification for the importanceof a 3D environment in the constructs that comprise the in vitro immunesystem.

The differences between 2D and 3D microenvironments include that:

(1) in 2D cultures, cells experience unnatural, anisotropic, externalcues from the artificial support, while in 3D cultures, cells are ableto migrate on the ECM in all dimensions;

(2) in 2D cultures, the support (e.g., plastic, glass) is far more rigidthan naturally occurring ECM;

(3) cells grown in 3D culture are more resistant to apoptosis, and

(4) proteins secreted by cells can better interact with and be organizedby a 3D culture surrounding ECM and influence cell behavior.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of immunology, histology, microbiology,cell and tissue culture, and molecular biology within the ordinary skillof the art. Such techniques are explained fully in the literature. Allpublications, patents, and patent applications cited herein areincorporated by reference in their entirety.

The design of the in vitro artificial immune system (AIS) of the presentinvention comprises:

1. three basic, functional, immunological tissues:

-   -   a. skin and/or mucosal equivalent (the vaccination site),    -   b. a lymphoid tissue equivalent (LTE (the lymph node), and    -   c. lymphatic and blood vascular network equivalents to connect        the other two tissues,

2. a cell source for reproducible and repeatable testing, and

3. a bioreactor to house and integrate the immunological tissues andrapidly assess and test vaccine efficacy.

The AIS of the present invention further comprises:

(a) an in vitro skin and/or mucosal-equivalent scaffold (vaccinationsite, VS) that facilitates trafficking of blood monocytes andnon-monocytic dendritic cell (DC) precursors and supports their naturalconversion into mature antigen presenting dendritic cells within theartificial skin 3D tissue-engineered construct;

(b) a lymphatic vessel-like pathway from the vaccination site to thelymphoid tissue equivalent (LTE) for mature DC migration and a bloodvessel-like pathway for monocyte migration into the vaccination site(VS);

(c) a lymphoid tissue equivalent in a tissue-engineered scaffold with astructure that mimics lymph node geometry and contains appropriate lymphnode cell types;

(d) the above constructs that are functionally equivalent tissueconstructs that exhibit comparable properties to endogenous tissues;

(e) integration of these immunological tissue constructs in a modularbioreactor system.

Design and construction of a 3D perfusion mesh, membrane, or gel-likestructure for the in vitro vaccination site (VS) is an important featureof the present invention. The VS provides an important part of the modelof vaccine action. As a stand-alone system for vaccine studies, itenables the dissection of differences in mechanism between differentvaccines, adjuvants, drugs, biologics, cosmetics, and immunotherapycandidates and thus helps in the refinement and improvement of thesesubstances.

The design and construction of a blood endothelium pathway for monocytemigration to the in vitro VS is also important. A lymphatic endotheliumpathway from the in vitro VS to the LTE for mature DC migration isprovided. A 3D model consisting of vascular and lymphatic endothelialcells that supports transendothelial trafficking of monocytes and otherDC precursors in a manner that recapitulates in vivo differentiation andmigratory functions for a vaccination site (e.g., skin equivalent) canbe used for testing cosmetics, anti-oxidants, possible skin irritants,and other chemicals.

The AIS enables quantitative measurement of T and B cell stimulation:

(a) through a venue in the LTE for DC, CD4+ T, CD8+ T and B cells tomeet in one place, to test whether a vaccine or immunotherapy promotesoptimal levels of T cell help (TH1 or TH2) to induce cytotoxic Tlymphocyte (CTL) and B cell responses;

(b) enabling DC, T and B cells to meet in a 3D environment withextracellular matrix and support cells that mimic the environment of thelymph node where the three cell types can interact;

(c) the inclusion of endothelium so that monocytes and DCs can interactwith endothelial cells during recruitment and emigration;

(d) the presence of a more representative population of cells and ofcells that can migrate across the endothelium and differentiate inresponse to local tissue signals (for example, to distinguish theeffects of TLR 9 (Toll-like receptor 9) ligands versus TLR-4 ligands, asthey are expressed differentially on multiple DC subtypes).

The present invention further comprises:

1. the use of novel biomolecule controlled-release strategies (such ascontrolled-release microspheres, direct injection nanosyringes, dualfunctionality nanogels, directed degradation rates);

2. the use of directed cell migration from ETCs to and from the vascularhighway using, for example, chemotaxis or the influence of shear forceson transendothelial migration (to orchestrate the cellular migratoryroutes to the VS (monocytes), from the VS (mature DCs), and into the LTE(mature DCs, T and B cells);

3. directed cell migration from ETCs to and from the vascular highwayusing magnetic microbead approaches (magnetic microbeads andelectromagnetic fields may also be used to move cells betweencompartments of the AIS);

4. the design and construction of a lymph node-like structure (the LTE)in a 3D scaffold with a structure that mimics lymph node geometry andcontains appropriate lymph node cell types, cytokines and chemokines;

5. facilitation of an approach to create tunable microenvironments tostudy initiation of the immune response via a model of the lymph node'sT zone in the LTE design (including engrafting T zone fibroblasticreticular cells (FRCs, stromal cells of the T zone) on ordered,macroporous, hydrogel scaffolds akin to inverse opal structures and theuse of both synthetic and natural extracellular matrix (ECM) materials,to achieve 3D structures that provide a physical structure mimicking thelymph node's “open” reticular network, containing lymphocytes andbiochemical cues (such adhesion motifs and chemokine gradients) expectedby lymphocytes in secondary lymphoid tissues);

6. in the LTE, providing the lymph node-like function of T cell help forB cell antibody production (for example, distinct T and B cell areaswithin the LTE can be designed by the combined action of digitalprinting (directing assembly of T and B cells within distinct zones) andby controlled release technology (using, for example, microspheresreleasing T and B cell attractants to maintain T and B cell areas,respectively);

7. to assist in self organization of the LTE, BLC (B lymphocytechemoattractant, MV10 kDa), MIP-3β (macrophage inflammatory protein-3β,CCL19), and SLC (secondary lymphoid tissue chemokine) chemokinemicrospheres for controlled release with the LTE matrix; additionally,microspheres may be co-printed with T and B cells into LTE scaffolds (inan alternative embodiment, microspheres can be directly encapsulatewithin the “struts” (e.g., using polycaprolactone) of the hydrogelmatrix during polymerization in a criss-cross pattern, much like a“Lincoln log”) (in still another embodiment, FRC-engrafted T cell areascan be used, assuming the stromal cells guide T cell localization withinscaffolds);

8. the use of a digital printing BioAssembly Tool (BAT) capable ofprecision-manufacturing 3D ETCs, specifically with fine volumetriccontrol to create 3D constructs;

9. use of an engineered, cellular microfluidic, environmental bioreactorthat can sustain multiple immunological ETCs and be used to mimic thehuman immune system;

10. use of a miniature 3D housing with internal channels through which anutrient-rich liquid is pumped to “feed” the immunological cells. Thewalls of these channels are modified to allow endothelial cellattachment, creating an artificial endothelium, or are fabricated from abiologically compatible material that does not alter cell behavior.Cells within the constructs rely on constant fluid flow, not just fornutrients, but also as a signal that all is well and that they shouldcontinue with their business via chemokines. Nutrient fluid primes thesystem before various cells are injected (via syringe initially); thecomplete AIS is then functionally connected to a pumping that simulatesblood flow for the nutrient/oxygen solution. In a preferred embodiment,a pulsed pumping mechanism is used to better mimic the situation seen inthe blood vasculature. Embodiments of miniature size and transparentarchitecture enable the visualization of the tissue construct componentsin situ under a microscope;

11. use of the AIS to test the efficacy of vaccine adjuvants, vaccineformulations, and immunotherapies in vitro for high throughput vaccineand immunomodulator screening in an ex vivo immune system with anappropriate repertoire of T and B cells;

12. the manufacture of monoclonal antibodies in the AIS by activating Bcells in the LTE.

In another embodiment of the LTE, adjacent T and B cell zones arecreated, thereby mimicking the natural separation of B and T zones in areal lymph node. In this embodiment, T and B zones of the LTE arecreated using microcarriers. Much is now known about the cultivation ofcells on microcarriers; these are particles typically about 100 to 5000μm in diameter, rough surfaced or porous, coated with the necessarycomponents of the extracellular matrix, on which a variety of anchoringdependent cells can grow and proliferate. The model system is akin toparticles in a box. Matrix materials for the microcarriers may includelymphoid tissue particulate ECM material, protasan, collagen,protasan/collagen mixes, PLGA (poly(lactide-co-glycolide)), and otherscaffold materials.

The general approach to creating such a LTE comprises:

1. loading microcarriers with appropriate adhesion ligands, such aschemokines, for the attachment of T and B cells; the microcarriers canbe natural or synthetic, dense or porous and of various sizes dependingon the desired packing density;

2. culturing s T and B cells on the microcarriers; the T and B cells canbe cultivated together (FIG. 1A), or cultivated separately on theirrespective microcarriers (FIGS. 1B and 1C);.

3. bringing together the T and B cell-populated microcarriers in contactin a porous container (akin to a “tennis ball basket;”); and

4. allowing the microcarriers to pack ‘intelligently;’ such packingdensity allows cell penetration.

FIG. 1D is a schematic showing binding T and B cells to themicrocarriers having CXC 13 ligand as an adhesion ligand. As shown inFIG. 1E, the size of the microcarrier particle influences the porosityand the openings between the microcarriers. In addition, the shape ofthe microcarriers (e.g., spherical, irregular shaped, etc.) also impactson the optimized packing densities.

It is also envisaged that the T cells could be “free” in media while theB cells are primarily located on microcarriers or alternatively that theB cells could be “free” in the media while the T cells are primarilylocated on the microcarriers as other variations on this theme.

The development of an in vitro immune system requires:

1. engineering of 3D scaffolds and cell differentiation cascades thatallow the formation of three basic, functional, biological tissues, inparticular, immunological tissues such as:

-   -   a. skin and/or mucosa (vaccination site, VS),    -   b. lymph node (lymphoid tissue equivalent, LTE), and    -   c. lymphatic and blood vascular network equivalents,

2. a cell source for reproducible and repeatable testing, and

3. a microfluidic bioreactor to house and integrate the immunologicaltissues.

How such a system works is illustrated schematically in FIG. 2.

There are sequential steps in the generation of an immune response to avaccine in a mammal. First, a vaccine acts on immune cells in the skin,gut, or mucosal site of vaccination to activate cells. Second, afterimmunization with a vaccine, dendritic cells (DCs) migrate out of thesite to the draining lymph node via lymphatic highways. Third, dendriticcells in the draining lymph node (or other secondary lymphoid tissues)interact with T and B cells to activate antigen specific lymphocytesthat are capable of activating further immune responses, eliminating thepathogen through multiple effector mechanisms and transforming intomemory cells with long term memory of antigen.

This three-step process is mimicked functionally and structurally in theAIS of the present invention. First, the antigen/pathogen acts on immunecells in the in vitro vaccination site (VS, e.g., skin equivalent ormucosal tissue equivalent) to activate antigen presenting cells andstart the maturation process. Second, as cytokines, chemokines, andchemicals are produced at the site of vaccination, dendritic cellsmigrate out of the site to the lymphoid tissue equivalent (LTE) vialymphatic vessels and complete their maturation process. Third,dendritic cells in the LTE interact with T and B cells to activateantigen-specific lymphocytes that are capable of activating furtherimmune responses, eliminating the pathogen through multiple effectormechanisms and transforming into memory cells with long-term memory ofantigen.

The AIS comprises three immunological tissue constructs corresponding tothe three basic steps in vaccine or immunotherapy action. Tofunctionally reproduce these three steps, the AIS comprises three tissueengineered constructs:

1. an in vitro VS scaffold that facilitates trafficking of bloodmonocytes and non-monocytic dendritic cell precursors and supports theirnatural conversion into mature antigen-presenting dendritic cells withinthe artificial skin 3D construct,

2. a lymphatic vessel-like pathway from the vaccination site (skinequivalent) to the lymphoid tissue equivalent for dendritic cellmigration; likewise, a blood vessel-like pathway for monocyte migrationto the vaccination site (skin equivalent and/or mucosal equivalent), and

3. a lymphoid tissue equivalent (LTE) in a scaffold with a structurethat mimics lymph node geometry and contains appropriate lymph node celltypes.

These functionally equivalent tissue constructs exhibit comparableproperties to endogenous tissues. These functionally equivalent tissueconstructs are integrated in a modular bioreactor. The AIS is designedto perform high throughput vaccine and immunomodulator screening in anex vivo immune system that provides the appropriate repertoire of T andB cells within a bioreactor system.

The Vaccination Site (VS)

The efficacy of a vaccine reflects, to a large degree, the quality ofthe initial interactions with cells at the site of vaccination (FIG. 3).Consequently, to create a useful model of vaccination, it is importantto construct an artificial vaccination site in vitro. Such a vaccinationsite will act as a skin-, gut-, or mucosal-equivalent tissue andcomprises a skin construct (or a mucosal tissue, such as lung), togetherwith vascular and lymphatic endothelium and blood-derived hematopoieticcells. The skin construct can be derived from many sources, includingcomplex sources, such as cadaveric human skin, less complex sources,such as commercially available skin-like products (EpiDerm, Episkin), orsimple skin-like structures (using many different preparations of ECMand sources of skin fibroblasts and keratinocytes) optimized forintegration into the in vitro system.

Blood cells (including monocytes) can be placed (or can flow) along thevascular endothelium. Such cells naturally migrate, convert to dendriticand other cells, and become resident in the skin.

If dendritic cells are present in the correct subtype and state ofmaturation for resting skin, the vaccination site is then ready toaccept a vaccine candidate for testing. Upon vaccination, the vaccinewill interact with skin-resident cells to induce further migration ofmonocytes and other cells into the skin, and their subsequentdifferentiation into more antigen-presenting cells (APCs), includingmacrophages and dendritic cells. Dendritic cells (DCs) and otherantigen-presenting cells (APCs) pick up vaccine antigen and will beinduced to migrate across the lymphatic endothelium to drain in thelymphoid tissue equivalent. DCs arriving in the LTE interact with T andB cells to initiate an adaptive immune response, and depending on thematuration state of the DCs, they will activate T and B cells todiffering extents.

In summary, together with the LTE (described below), the vaccinationsite provides an important model of vaccine, chemical, adjuvant, drug,or biologic action. However, even without the LTE, the vaccination siteis an important stand-alone model for vaccine studies that enables thedissection of differences in mechanism between different vaccines orchemical candidates and thus helps in the refinement and improvement ofvaccines. It is an important stand-alone model for testing cosmetics,fragrances, antioxidants, possible skin irritants, and other chemicals.

The In Vitro Lymphoid Tissue Equivalent (LTE)

The ultimate output of a vaccine occurs in the lymphoid tissues, whereantigen-specific T and B cells are activated and partly convert tomemory cells that have been notoriously difficult to detect in vitro. Tomimic a natural immune response in vitro, it is therefore essential tobuild a lymphoid tissue equivalent (FIG. 4) and connect it up with thevaccination site via lymphatic vessels. In vivo, vaccine-derived antigenis transported to lymph nodes by diffusion along lymphatic vessels tolymph node cells, or by migration of mature DCs that have internalizedthe antigen, to the draining lymph node. In the lymph nodes, DCsactivate antigen-specific T cells and, in conjunction with helper Tcells, help to activate antigen-specific B cells to elicit an immuneresponse.

The strength and quality of the T and B cell responses depend on theamount of antigen delivered and on the subtype and maturation state ofthe DC (APC) carrying the vaccine-derived antigen. Two- and three-wayinteractions between the key cells (dendritic cells, B and T cells)occur in spatially segregated regions of the lymph nodes in a sequentialorder of events. To simulate this process, an artificial lymphoid tissueor lymphoid tissue equivalent (FIG. 4) can be constructed with lymphnode-like geometries and spatial organization in vitro using acombination of tissue engineering, materials science, and biologicalstudies. For example, immune cells are highly responsive to chemokinegradients, and thus the design of scaffolds containing organizedgradients of these signaling molecules allows the synthetic lymph nodetissue to self-organize, in a fashion similar to that in native tissue.The formation of native tissue can also be studied in parallel touncover further molecules to help form in vitro-organized tissues. Suchcomplex synthetic structures can also be fabricated using the digitalprinting BioAssembly Tool (BAT).

In an embodiment of the present invention, once the LTE is assembled, itis also possible to use it as a “biofactory,” biosynthesizing variousdesired biomolecules (such as cytokines, proteins, antibodies). Forexample, if an antigen is presented to B cells, they can createantibodies in the LTE. Potentially, the created antibodies could also bemonoclonal, depending on the repertoire of B cells and how the peptideis presented to the B cells.

In Vitro Lymphatic and Blood Vascular Highways

The present invention provides designs for endothelium pathways (e.g.,using different matrix formulations, sources of endothelial cells, andgrowth conditions) that facilitate cell immigration and emigration intoand from the VS and the LTE, as well as between the VS and LTE. Aschematic of the in vitro artificial immune system ensemble is shown inFIG. 5. The artificial immune system can have a general bioreactordesign that is mechanistically different than the natural immune system,though similar in terms of functionality. In a preferred embodiment, thethree immunological ETCs are integrated in a miniature, engineered,cellular environmental bioreactor. This design uses two functionallyequivalent membranes in a sequential order to create a functional VS andlocalized collections of T and B cells on or around particles, tofunction as the LTE Important design considerations are to emulatebiological functions, minimize media volume between zones to increaseefficiency of cell trafficking, and provide a means of evaluatingantigenic responses. By integrating and minimizing the media volume,potential for cell migration within and between the immunological ETCsis dramatically enhanced and can provide an increased immunologicalresponse.

However, it is not necessary to have the VS and LTE in an integratedbioreactor. In an alternative embodiment, mature DCs from the VS can bephysically positioned in the LTE. These mature DCs will activate T cellswithin the T cell zones and B cells within the B cell zones of the LTE.Thus, it will be possible to test and characterize both the VS and LTEand the interactions between the mature DCs from the VS and the T cellsin the LTE in a non-integrated fashion.

The general, basic cascade of events for AIS operation is as follows:

monocytes and other blood derived cells (PBMCs) are injected into theblood vascular highway;

chemokines (either natural to the VS or intentionally added) attractmonocytes to enter into the VS;

monocytes differentiate into immature DCs (iDCs); iDCs mature inresponse to vaccination in the VS;

chemokines attract mature DCs into the lymphatic highway;

chemokines (either natural to the LTE or intentionally added) attractmature DCs into the LTE; and

mature DCs in the LTE activate T and B cells.

Monocytes and dendritic cells will naturally interact and migrate acrossthe vascular and lymphatic endothelia. In other embodiments chemokinescan be used to direct the migration of the cells, as can magneticmicrobeads. Magnetic beads together with miniaturized electromagnets area convenient mechanism for manipulation of cells in a bioreactor. Forexample, cells with appropriate surface markers (receptors, epitopes)can be selected using the beads and selected cells can be transportedfrom one local environment to another, bringing cells in contact with,e.g., desired surfaces, environments, or other cells (see Examples).

Universal Cell Source

The in vitro AIS device would at a minimum have to contain T cells, Bcells, and antigen-presenting cells, but would preferably include othercellular components, such as endothelial cells to create theendothelium, neutrophils and mast cells to respond to vaccine-derivedsignals, fibroblasts cells that mediate initial entry of a specificpathogen into the skin, or cells from target organs (e.g., lung) thatthe pathogen in question infects. The T and B cells would be locatedprimarily in the LTE, the monocytes/DC precursors in the bloodvasculature and the vaccination site, and the blood and lymphaticendothelial cells would be in the blood and lymphatic highways,respectively.

In an embodiment, the immune cells from peripheral blood mononuclearcells (PBMC) will be from individuals who are HLA (human leukocyteantigen)-matched to the endothelium and VS matrix cells used in thesystem. Peripheral blood mononuclear cells represent a heterogeneouspopulation of immune cells (T cells, B cells and various granulocytes)that arise from pluripotent hematopoietic stem cells in the bone marrow(Janeway, et al., Immuno. Biology (1999), Garland Publishing/ElsevierLondon, UK). In an alternative embodiment, using stem cells, it may bepossible to provide all the necessary cell types for the system. Instill another embodiment, parallel with progenitor cell development,cells from a humanized mouse node can be used to initially populate thevarious tissue constructs.

The Bioreactor

In the integrated AIS bioreactor, a nutrient-rich liquid is pumpedthrough internal channels in a 3D housing to ‘feed’ the immunologicalcells. The walls of these channels are modified to allow endothelialcell attachment, creating an endothelium, or are fabricated from abiologically compatible material that does not alter cell behavior.

To overcome obstacles in developing the AIS bioreactor, in oneembodiment, laser micromachining with ultra-short pulse lasers can beused to design and fabricate the channels so that the fluid flows well.In other embodiments, micro-stamping, laminates, or standard CNC andother milling processes can be used.

Cells within the constructs will rely on a constant flow, not just fornutrients, but also as a signal that all is well and that they shouldcontinue with their business via chemokines. Nutrient fluid will primethe system before various cells are injected via syringe initially orusing the cell sorting systems described.

The complete artificial immune system is then connected to a pump thatsimulates blood flow for the nutrient/oxygen solution. In a preferredembodiment, the pumping mechanism can also be pulsed, to better mimicthe blood vasculature. The entire assembly can then be inserted into anincubator that regulates temperature, humidity, and concentrations ofoxygen and carbon dioxide to best simulate the natural in vivoenvironment.

In a preferred embodiment, the bioreactor system can be constructed tobe of the order of a few inches in total size, potentially allowing thein vitro immune system bioreactor apparatus to be built into otherstationary and portable analytical instruments. Embodiments of miniaturesize and optical transparency allow viewing of the tissue constructcomponents in situ using a microscope.

Using the AIS, it is possible to rapidly test and evaluate the immuneresponse to vaccines and other substances. Several concepts arepresented to organize the tissue and activate it appropriately toreceive vaccines. In one embodiment, the integrated engineered tissueconstructs incorporate chemotaxis and engineered-release microparticlesto allow control of temporal, spatial, and dose parameters of variousbiomolecules for tissue and cell assemblage and programming In anotherembodiment, constructs provide an environment that enables the stromaand parenchyma to self-assemble into a native-like tissue viacommunication achieved through cell-cell, cell-matrix, structural andendogenous growth factor cues that the cells themselves create; noexogenous growth factors may be necessary to induce given phenotypes.

EXAMPLES Example 1 In vitro and In vivo Control of Chemotaxis of BothiDCs and Monocytes using Controlled-release Microparticles

PLGA (poly(lactide-co-glycolide)) microspheres provide steady controlledrelease of encapsulated chemokines. FIGS. 6A shows example micrographsof microspheres encapsulating fluorochrome-labeled fMLP chemokine (amonocyte and immature DC chemoattractant). FIG. 6B shows the releasekinetics for both low molecular weight peptide chemokines and a 10 kDachemoattractant, MIP-3β (macrophage inflammatory protein-3(3). As shownin FIGS. 7A-7D, human monocytes and dendritic cells move towardsmicrospheres releasing the chemoattractant fMLP(N-formyl-methionyl-leucyl-phenylalanine) in an in vitro setting. FIG. 8shows in vivo mouse immunohistochemical staining for monocytes that wereattracted to the chemokine MIP-3β in an implanted extracellular matrixscaffold. Synthetic inverse opal hydrogel scaffolds were synthesizedthat support T-cell migration and interaction with B cells (FIG. 8A),and support attachment and growth of high cell densities, which is animportant feature in mimicking the microenvironment of the lymph node(FIG. 8B).

Example 2 Designer Scaffold Structures

Designer scaffold structures were constructed to test cell viability,cell motility, and nutrient flow for bioreactors and have studied cellmotility as a function of construct stability for collagen gels. FIG. 64shows HUVEC cells growing on protasan/collagen matrix on a nylon mesh.High magnification SEM of the nylon membrane and interspersedProtasan/collagen matrix material is shown in the top image. Seeding ofthe primary layer of HUVEC cells was accomplished on an invertedmembrane (left, Side 1), then 24 hours later, brought to an uprightposition (right, Side 2) where the second layer was applied. Phasecontrast images of each plane of HUVEC cells is shown in the center twolower images, with the left being the first layer, and the right beingthe second layer applied.

Example 3 Digital Printing Technology

Preliminary hardware and software ETC heterogeneity digital printingprototypes have been developed. FIG. 9 shows the mockup of a digitallyprinted lymph node and a retinal image of vasculature. This mockup lymphnode comprises six biocompatible hydrogel layers, four differentpatterns, and three materials. The vasculature image has been built withmultiple layers of biodegradable construction material with featuresizes that range from 100 to 3,000 μm. The objects were fabricated withthree dispensing nozzles each.

Example 4 3D Tissue Constructs

3D biology is important to induce proper functionality of theimmunological ETCs. An important approach to studying cellular processesis to culture cells in vitro. This has typically involved plating cellson plastic or glass supports. In this application, cells grown on solidor filter support are referred to as two-dimensional (2D) cultures. Such2D cultures on porous supports have been useful for studying manyaspects of biology. However, much more in vivo-like conditions can nowbe realized in 3D cultures.

The majority of vaccines are delivered via the skin or mucosal surfacesof the body. Within the delivery site, key steps in the action ofvaccines are the differentiation of precursor cells to dendritic cells(DCs), the acquisition of antigen by DCs, and the maturation of the DCsto optimally process antigen and activate T cells, B cells, and otherimmune cells. During the period of DC maturation, DCs must also bemobilized and transported to a position within the T cell zone of lymphnodes where they can optimally encounter and select the T cells with themost appropriate T cell receptor to respond to the processed antigen inquestion. In some cases, antigens diffuse to the draining lymph nodedirectly, and are then captured by lymph node DCs.

To date, there has been no model of these early steps of vaccine actionin humans; furthermore, it is not possible to study these steps inhumans because it occurs in inaccessible peripheral tissues and not inthe more accessible blood. Therefore, the present invention provides amodel system that enables studies of these early steps in a morerealistic context of cells and structures than is currently available.

The present invention provides a model vaccination site in vitro fortesting the efficacy of vaccines in antigen loading and activation ofimmune cells, especially dendritic cells. The vaccination site comprisesimmune cells, including monocytes and dendritic cells, embedded in askin-equivalent (or mucosal tissue equivalent) tissue that is attachedto vascular and lymphatic endothelium. This in vitro tissue constructenables rapid testing of vaccine candidates and evaluation of theireffects at the early steps of vaccination. The vaccination site is thenintegrated with the lymphoid-tissue equivalent to form an artificialimmune system for testing vaccine efficacy in a more complete model ofthe human immune system.

The VS can be envisaged as a skin equivalent or a vaccination siteequivalent to the in vitro immune system. In an alternative embodiment,a mucosal tissue equivalent can also be readily envisaged. The skinequivalent is preferred because of recent advances in skin tissue modelsin vitro. Indeed, a number of studies exist in the literature that haveused human skin explants and still others have probed DC behavior involunteer subjects, who consented for biopsies during immune modulationstudies (Cumberbatch, et al., Br. J. Dermatol. 141:192-200, (1999)).There have been a few reports on the integration of DCs in skin in vitrocultures (Regnier et al., J. Invest. Dermatol. 109:510-512, (1997);Fransson, et al., Br. J. Dermatol. 139:598-604, (1998)). Finally andmost importantly, skin is the most common site of vaccination.Consequently, it is logical to use skin models to study the early stagesof DC activation in the periphery.

The present invention provides a reproducible skin equivalent model fortesting vaccine candidates and other drugs, biologics, and chemicals andfor integrating this tissue with a lymphoid tissue equivalent in vitroto measure T and B cell immune responses. A step-wise approach isprovided to build a 3D structure that comprises vascular and lymphaticendothelial cells that can support transendothelial trafficking ofmonocytes and other DC precursors in a manner that recapitulates in vivodifferentiation, maturation and migratory functions.

It is known that a 3D tissue construct that permits heterologouscell-cell interactions impacts the differentiation of DC precursors,including monocytes, in a manner that more closely mimics an intacthuman system than is observed in 2D culture (See, e.g., Edelman &Keefer, Exp. Neurol. 192:1-6 (2005)). Specifically, co-culture of wholePBMCs with vascular endothelial monolayers, grown on eitherreconstituted type I collagen matrices (Randolph, et al., Blood 92:4167-4177 (1998a); Randolph, et al., Science 282:480-483 (1998b);Randolph, et al., Proc. Natl. Acad. Sci. USA 95:6924-6929 (1998c);Randolph, et al., J. Exp. Med. 196:517-527 (2002)) (FIG. 10) or nativeamniotic connective tissue (Randolph & Furie, J. Exp. Med. 183:451-462(1996)) promotes the passage particularly of monocytes across theendothelium, largely in response to endogenous production of thechemoattractant monocyte chemoattractant protein (MCP)-1 (CCL2)(Randolph & Furie, J. Immunol. 155:3610-3618 (1995)). This is consistentwith the knowledge that many monocytes leave the blood each day, undernormal steady state conditions. When the endothelium is activated, otherinflammatory cell types, such as neutrophils, can traverse theendothelium, again with the same regulatory events that are understoodto operate in vivo (Furie & McHugh, J. Immunol. 143:3309-3317 (1989)).If the fate of monocytes is followed with time in endothelialcell/collagen cultures, it becomes apparent that a substantial fractionof monocytes increase production of a range of molecules (including MHCII, CD40, CD83, CD86) known to be upregulated in DCs and these cellsalso acquire migratory properties such that they migrate out of thecultures, crossing the endothelium in the ablumenal to lumenaldirection, away from the vascular endothelium and away from themacrophages that remain resident in the subendothelial matrix.

As shown in FIG. 10A, vascular endothelial cells grown on 3D constructsof fibronectin-coated collagen form intercellular junctions that remainintact after passage of monocytes into subendothelial matrix toincreasing depths (arrowheads, monocytes visualized by differentialinterference contrast microscopy). En face views and a cross-section ofthe cultures are shown, where emigrated leukocytes are distributedthroughout the matrix under the characteristically flat endothelialmonolayer. As described in design features 1 and 2, a lymphaticendothelial monolayer or an epidermal monolayer, respectively, on thecurrently bare lower surface of such a matrix. FIG. 10B is a schematicdiagram showing the stages of monocyte behavior in such a 3D culture.The image on the left depicts the sequence of observations when thematrix does not contain a source of microbial antigen, whereas theimages on the right depict the sequence of observations made when yeastparticles (zymosan) are incorporated as a model microbial antigen in thematrix. In stage I, incubation of peripheral blood mononuclear cells(PBMCs) are incubated with endothelium for 1.5 hours results in thetransmigration of most monocytes (3), some BDCA1+ blood dendritic cells(data not shown), natural killer cells (Berman et al., J. Immunol.156:1515-1524, (1996)), but few lymphocytes, into the subendothelialcollagen. Of the few lymphocytes that do migrate, these are likely of amemory phenotype (Gergel & Furie, Infect. Immun. 69:2190-2197, (2001)),consistent with our understanding that naive T cells traffic into lymphnodes directly and memory T cells can enter tissues. In stage II, thecell culture is washed, and monocytes accumulated in the subendothelialmatrix are left with an intact endothelial monolayer, where themonocytes engulf phagocytic particles if such particles have beenincluded in the collagen matrix. In stage III, some of the phagocyticmonocyte-derived cells retraverse the same endothelium and accumulate inthe apical compartment. These reverse-transmigrated monocytes previouslyor simultaneously differentiate into DC. Photographs (upper right, B)show their characteristic morphology. When no activation stimuli areincluded in the cultures (left), the reverse-transmigrated cells areimmature DCs and promote T cells to produce IL-10 as observed byintracellular cytokine staining. Many of these cells are non-adherent,like DCs, but a few spreading cells are similar to less differentiatedmonocytes (left photo inset, B). When activation stimuli are included inthe cultures, the reverse-transmigrated cells become mature DCs andpromote development mainly of IFNγ producing T cells. C) Monocytes canbe infected with influenza to measure activation of IFNγ induction andexpansion during recall responses in T cells from adults previouslyinfected with flu (Qu, et al., J. Immunol. 170:1010-1018, (2003)). Thenumber of T cell clones that begin in proliferate (each represents a“spot” in ELISPOT assay) in response to presentation of the processedvirus is more than 3-fold increased when monocytes are permitted todifferentiate in the 3-D endothelial cultures (filled bar) compared withtheir response when they are cultured on bare plastic. Even in theabsence of a maturation stimulus in the endothelial/collagen cultures,some monocytes accumulate MHC II in perinuclear compartments, indicativeof immature DCs (Mellman, et al., Cell 106:255-258, (2001)), in contrastto the same monocytes cultured on plastic where they express a greatlyreduced amount of MHC II on the cell surface, more characteristic ofmacrophages (C, photos).

As DCs are known to traffic substantially through lymphatic vessels, bytraversing lymphatic endothelium in the ablumenal to lumenal direction,these data demonstrate that the in vitro model mimics aspects of DCtrafficking via lymphatics. Experiments identifying molecules thatmediated DC migration in this model and then evaluating whether the samemediators control DC migration in intact authentic human skin explants,support this (Randolph, et al., Science 282:480-483 (1998b); Robbiani,et al., Cell 103:757-768 (2000)). This model system has also, forexample, allowed examination of the role of monocyte heterogeneity indifferentiation to DCs; the CD16+ monocyte subset preferentiallydevelops into DCs over other monocytes (Randolph, et al., J. Exp. Med.196:517-527 (2002)). Recent in vivo studies in mouse have identified apopulation of monocytes apparently equivalent to CD16+ monocytes, andstudies indicate that this subset readily becomes DCs (Randolph, et al.,J. Exp. Med. 196:517-527 (2002)). Thus, the 3D model of the presentinvention mimics normal immunophysiology.

Before microbes can be engulfed and destroyed, leukocytes in theperiphery must be able to reach them. The process is a complex one, andmutational data indicate that it is very important: severeimmunodeficiencies result from a failure of leukocyte adhesion,diapedesis, and chemotaxis, which have not been addressed in skinequivalents thus far.

By increasing in a step wise fashion the complexity of the 3D construct,and conducting assays for verification along the way, a faithfulrecapitulation of the events that regulate recruitment of DC precursorsand other inflammatory cells that would modulate their responses isachieved. The differentiation of these DCs in response to vaccineformulations or characterized antigens/pathogens, and their traffickinginto lymphatic vessels. In one embodiment, this can be achieved usingprinted scaffolds and the novel matrices/methodology described herein,and additional experience in the isolation and growth of skin derivedblood and particularly lymphatic endothelium (Podgrabinska, et al.,Proc. Natl. Acad. Sci. USA 99:16069-16074 (2002)). Moreover, our workwith human skin explants provides assays and a sound basis forcomparison of outcomes between the in vitro model and the behavior ofDCs in intact skin (Randolph, et al., Proc. Natl. Acad. Sci. USA95:6924-6929 (1998c)).

In one embodiment, a 3D model comprising vascular and lymphaticendothelial cells was constructed. The vascular and lymphaticendothelial cells support transendothelial trafficking of monocytes andother DC precursors in a manner that recapitulates in vivodifferentiation and migratory functions. As it is now possible todifferentially isolate vascular and lymphatic endothelium (Podgrabinska,et al., Proc. Natl. Acad. Sci. USA, 99:16069-16074 (2002)) and given theknowledge and resources for preparing these cells, a functional modelwas designed as diagrammed in FIG. 11. Several matrices can be used,including xenographic ECM sheets, natively polymerized human amnioticconnective tissue (Randolph & Furie, J. Exp. Med. 183:451-462 (1996)),reconstituted collagen matrices, protasan/collagen membrane scaffolds,or preferably matrices that contain fibroblasts and/or mast cells.Several commercial preparations of dermal tissues containing fibroblastsare available and these are readily prepared in vitro, for example byseeding fibroblasts with matrix components and allowing the fibroblaststo modify and contract these components, as described earlier.

It is anticipated that the process of incorporating cells within thematrix could be adapted for the incorporation of a variety of cells suchas fibroblasts or mast cells. In a preferred embodiment, vascular andendothelial monolayers are constructed that mimic the normal physiologyof these vessels in coordinating recruitment and trafficking of immunecells during immunization. In another embodiment, the endothelium can bederived from human foreskin (Podgrabinska, et al., Proc. Natl. Acad.Sci. USA, 99:16069-16074 (2002)) or from adult skin.

Using a 3D in vitro model, DC migration, an important process in theinitiation of immunologic priming, has been examined (Podgrabinska, etal., Supra, (2002)) Robust translation between the in vitro constructs,ex vivo models, and in vivo studies can be made. In an initial screen ofa large panel of neutralizing mAbs, it was found that mAbs thatrecognized ABCB 1 not only blocked reverse transmigration ofmonocyte-derived cells in the in vitro endothelial cell/collagenconstructs, but the same mAbs also effectively prevented the migrationof Langerhans cells from human epidermis. Further analysis of thisfamily of lipid transporters revealed strong expression of anothermember ABCC1 (ATP-binding cassette protein C1, MRP-1) in human skin DCs,and specific antagonists of ABCC1 also block skin Langerhans cellmigration from explants. In mice, ABCC1 is much more strongly expressedthan ABCB1. Functional studies indicated that ABCC1 participates in DCmigration in vivo. Thus, the in vitro model in which human monocytesbecome DCs in conjunction with their passage through endothelialmonolayers proved useful as a screening tool for relevant mediators ofmigration. In a similar experiment, an important role for chemokinereceptor CCR8 in reverse transmigration in vitro and then subsequentlyin vivo has been revealed (Qu, et al., JEM 200:1231-1241 (2004)).

Thus, the differentiation and migration of DCs in the in vitro model ofthe present invention accurately reflects outcomes in vivo, even aftercrossing the human-mouse species barrier.

The advancements set out in the present invention over previousmethodologies include, as a result of this design feature, more naturalrecapitulation of lymphatic trafficking. The monocyte-derived DCs aredesigned to be redirected to migrate across the lymphatic endotheliumafter first traversing the vascular endothelium. Based on studies ofendothelium in 2D cultures, these features will be accurately maintained(Podgrabinska, et al., Proc. Natl. Acad. Sci. USA, 99:16069-16074(2002)).

Example 5 Loading VS with Leucocytes

Further probing of the veracity of the VS design can be conducted using,for example, immune stimulation assays. Antigens of differentcompositions can be used, including stimuli delivered by variousdelivery depots. If the system operates as in vivo, a greater variety ofleukocytes, including granulocytes will cross the vascular endotheliumand enter the matrix after introduction of a pathogenic stimulus, likebacteria, compared with cultures not activated with such a stimulus.Viruses may promote a cellular infiltration of a different composition.It is expected that primarily DCs and memory T cells will traverse thefull construct, crossing first vascular endothelium, penetrating theconnective tissue matrix where they may acquire deposited antigen, andthen trafficking across the lymphatic endothelium.

In one embodiment, the cells could be adult white blood cells and adulthuman skin derived vascular and endothelial cells. In such anembodiment, there would not be histocompatibility between endothelialcells and the leukocytes. In another embodiment, histocompatibility maybe achieved by using all cells derived from human embryonic stem cells(ESCs). It is presently feasible to ensure that data generated in themodel using cells from different donors is similar to that in a fullyhistocompatible system.

In another embodiment, full histocompatibility of leukocytes andendothelial cells can be achieved by working with endothelial cellderived from a particular male donor's foreskin and using cord bloodfrom the same individual as the donor for leukocytes, including themonocytes and T cells. Foreskin tissue and cord blood can be obtainedfrom the same donor (Podgrabinska, et al., Supra (2002)). Additionally,cord blood is a useful source of monocytes and T cells that generallyrecapitulates the trafficking patterns and generation of DCs observedwith adult monocytes (Qu, et al., J. Immunol. 170:1010-1018 (2003)) andthe use of cord blood monocytes can lead to priming of the autologous Tcell population. In another embodiment, HLA-matched foreskins and cordblood can be used to achieve histocompatibility.

Example 6 Fabrication of VS Scaffold using Protasan

In another embodiment of the present invention, porous chitosan/collagenscaffolds are used in the vaccination site tissue-engineered construct.The membrane scaffold for the VS comprises a porous membrane comprisingpreferably natural biopolymers that accommodate confluent cultures ofvascular and lymphatic endothelial cells, and remain sufficientlypermeable to provide transmigration of monocytes during theirtransformation into immature dendritic cells.

In this embodiment, a VS scaffold was prepared by freezing, alkalinegelation, and vacuum drying. Briefly, Protasan (8 mg/ml) was depositedon a nylon mesh strainer with 70 μm pore size, then frozen slowly at−30° C. and placed in cold ethanol/NaOH solution (1 part saturatedNaOH+50 parts 95% ethanol) at −30° C., overnight. The strainer was thentransferred to pure, cold ethanol (−30° C.) and washed for ½ hr withoccasional stirring, and finally vacuum-dried. A picture of the scaffoldis shown in FIG. 12.

Example 7 Fabrication of VS Scaffold using Rat Tail Type I Collagen

In this embodiment, the VS scaffold was prepared by leaching, alkalinegelation, and vacuum drying. Protasan (0.5 mg/ml) plus rat tail type Icollagen (3.6 mg/ml) were placed in a 1.5 ml microfuge vial (200 μlcollagen (3.8 mg/ml) plus ˜5 μl 2% Protasan). Dry polystyrene beads (7μm size) were added 1:1 by weight to get a paste that was centrifuged at5000 g for 2-3 min The pellet was deposited sparingly on 100 μm poresize nylon mesh and air-dried at 60° C. The mesh was then placed inethanol/NaOH solution at room temperature for 2 hr with slow stirring.It was then washed in pure ethanol for half hour with slow stirring.Finally, the mesh was transferred into tetrahydrofuran (THF) for 1 hrwith slow stirring; it was then washed in pure ethanol and vacuum-dried.A picture of the scaffold is shown in FIG. 13.

Example 8 Fabrication of VS Scaffold using Bovine type I Collagen

In this example, the VS scaffold comprises a continuous collagenmembrane (a bovine type I collagen matrix deposited and congealed on thenylon mesh). Specifically, acidic bovine collagen (3 mg/ml) wasneutralized with sodium hydroxide (NaOH) on ice and deposited onto a 100μm pore size nylon mesh, laminated in a stainless steel O-ring so thatit could be accommodated in a bioreactor. The scaffold was congealed at37° C., 95% RH and placed in cell culture medium. A picture of thescaffold is shown in FIG. 14.

Example 9 Two-Side Culture of Human Endothelial Cells onProtasan/Collagen Porous Matrix

In this embodiment, confluent endothelial cells were grown on the VSmembrane matrix. Freshly expanded human vascular endothelial cells(HUVEC) were deposited on the bottom side of the nylon mesh strainercomprising a porous Protasan/collagen membrane. For this step, thestrainer was placed upside down in a culture well. The cell suspensioncontained ˜5×10⁵ cells/ml. After letting the cells anchor andaccommodate, another deposition of HUVEC was made on the oppositesurface of the membrane, with the strainer turned in its normalposition. The two-side culture was maintained in DMEM media for 12 days.Pictures of the scaffold are shown in FIG. 15.

Example 10 Permeability of the Two-Sided HUVEC Culture

The permeability of the two-sided HUVEC culture to peripheral bloodmonocytes was examined. A specimen of the two-sided HUVEC culture grownon a Protasan/collagen porous matrix was seeded with human PBMC, withand without the monocyte-specific chemokine MCP-1 placed underneath.FIG. 16 shows monocytes on the bottom of the chamber without MCP-1 (leftpanel) with MCP-1 (right panel), 30 min after application onto themembrane.

Example 11 HUVEC Culture Grown on a Bovine Collagen Membrane

Confluent HUVEC culture grown on a bovine collagen membrane supported bya nylon mesh. Cells demonstrated well-defined multi-angular morphologyand clearly visible intercellular contacts, characteristic of successful(“happy”) endothelial culture. Under proper seeding conditions,confluency was achieved in 24 hours (FIG. 17).

Example 12 Human Monocytes Permeates the HUVEC Culture

Human monocytes permeated the HUVEC culture on a collagen mesh-supportedmatrix (FIG. 18). 1.5 hour after depositing human PBMCs over the HUVECmonolayer (5×10⁶/ml), a high number of monocytes enter into the collagencushion, traversing the monolayer in the luminal-to-abluminal direction.The figure shows toluidine blue-stained cells in the collagen attachedto the ring mesh with cells 20 μm below the surface HUVEC cells.

Example 13 Construction of VS using Skin Epithelium

In the AIS of the present invention, skin epithelium is integrated intothe 3D tissue construct so that DC precursors can take up residence inthe epidermis and normal immunophysiology is maintained. In thisembodiment, the complexity of the vaccination site is increased toinclude key elements of the skin is based on the fact that skin is themost common site of immunization. Indeed, some of the latest vaccinecandidates that appear promising are actually skin patches applied tothe epidermis.

A good source of skin is freshly isolated cadaveric skin from consentingorgan donors. This split thickness skin is sufficiently functional tosupport migration of DCs through dermal lymphatics (Lukas, et al., J.Invest. Dermatol. 106:1293-1299 (1996)) and it has previously been usedto verify and identify novel mediators of DC migration from theepidermis (Randolph, et al., Proc. Natl. Acad. Sci. USA, 95:6924-6929(1998c); Robbiani, et al., Cell, 103:757-768 (2000)). Thus, authenticskin can be used to compare with the model of skin of the presentinvention; explants for sources of keratinocytes, fibroblasts,endothelium, DCs, and dermal matrix can also be used (FIG. 11).

Example 14 Construction of VS

The integration of Langerhans cells in “skin equivalents” has beenaddressed (Regnier, et al., J. Invest. Dermatol. 109:510-512 (1997);Fransson, et al., Br. J. Dermatol. 139:598-604 (1998)). These areencouraging descriptions, as one of the models showed that keratinocytescould support Langerhans cell differentiation from CD34+ progenitorsnaturally without the addition of exogenous cytokines (Frans son, etal., Supra (1998)).

In another embodiment of the present invention, epidermal cells aregrown at the air interface, to permit stratification of the variousnormal layers of skin. On the other side of the matrix vascularendothelial cells are cultured. At a later time, adult PBMCs or cordblood PBMCs enriched in CD34+ cells are applied to assess whethermonocytes or other DC precursors were recruited not only across theendothelium, but whether these cells migrate across the matrix, as iftraversing the dermis, and then move into the epidermal layer to occupyit with integrated Langerhans cells. If integration is observed, theintegrated cells can be retrieved to determine whether they acquiredfeatures specific to Langerhans cells, such as the Birbeck granule.Keratinocytes can also be seeded on the undersurface of a matrix withstrong tensile strength. Matrices with such strength are readilystretched across various templates, such as Teflon rings (Randolph &Furie, J. Immunol. 155:3610-3618 (1995)). These Teflon rings have beenfurther designed so that the stretched matrix provides a “floor” to aculture well.

Creating insert supports for both synthetic and natural membranes hasbeen accomplished using laminates, crimped rings, and adhesives.Laminates and adhesives have primarily been used to support polymermeshes, which in turn are provide mechanical strength to syntheticallyformulated biological membranes. Fabrication using the laminate processcomprises sandwiching a stretched mesh between two pieces of polymerlaminate, which are then sealed together, e.g., thermally. The adhesivemethod consists of stretching a mesh support and adhering a ring(comprising, e.g., stainless steel) using a biocompatible glue. Thecrimping method is shown in FIG. 53; in this, the membrane is compressedbetween two rings of suitable material, e.g., stainless steel.Generally, the laminate and adhesive methods are preferred for syntheticmesh supported membranes, while the crimping method can accommodate bothnatural, biological membranes and synthetic meshes. FIG. 19 showspictures of membranes crimped in between thin stainless steel rings.Using the crimping method, biological membranes can be supported withoutthe use of adhesives and pressed into a disk with thickness profile ofabout 400 μm or less. Epithelium is then grown on the topside orunderside of the matrix, which can be left at the air interface bysetting the structure on an inert, porous “stand” placed inside a tissueculture dish. Endothelium is then grown on the other side of the matrixto form a monolayer on the inner surface of the culture ring, to whichculture medium can be added (FIG. 11, right panel).

FIG. 65 is a picture of ring structures showing variable methods ofattachment of membranes for VS in the bioreactor. The left panel showsthe spiked ring design used to hold ‘wet’ membrane structures such asamniotic or UBM naturally occurring ECM membranes. The right panel showsthree methods used to attach ‘dry’ synthetic membranes to the ringstructure. Top left (next to the left side of the dime) is crimped,bottom left is by laminating the membrane between two rings of the samematerial, and bottom right (below the dime) is glued. A variety ofbiologically cell-friendly cyanoacrylates, epoxies, and silicones havebeen successfully used in attaching meshes to the rings. FIG. 66 showsHUVEC cells on the culture plate with a bead of Devon two-part epoxyapplied and polymerized in place prior to seeding. No ill affects on thecells were observed after 72 hours and cells reached confluence after 48hours.

Because endothelial cells could be grown on human amniotic connectivetissue, an acellular natively polymerized human connective tissue, in anoninflamed setting (Furie & McHugh, J. Immunol. 143:3309-3317 (1989)),amniotic matrices were prepared from human placenta. Vascularendothelium were grown on both sides of the amniotic matrix (FIG. 47)without underlying inflammatory signals, because on this matrix,neutrophils could be added without being recruited across theendothelium (only about 1% of neutrophils added migrated in thesecultures, within the range considered to be noninflammatory (Furie &McHugh, J. Immunol. 143: 3309-3317 (1989)). A positive control for“inflammation” in these experiments was to stimulate some of theendothelial/amnion constructs with the pro-inflammatory cytokine, IL-1an average of 76% of neutrophils migrated in the same experiments withIL-1, where only 1% migrated in its absence) (FIG. 47). These resultsdemonstrate: (a) construction of a culture system that supported growthof two layers of endothelium and (b) construction of culture system thatis not inherently inflammatory, but which can become so in response toappropriate stimuli (e.g., IL-1).

In addition to having endothelial layers to control recruitment into andout of the vaccination site, other cell types can be added to thecultures (e.g., fibroblasts; they are a normal component of alltissues). The isolated and cultured primary human fibroblasts wereobtained from human placenta. These cells were seeded in the matrixafter the addition of the first endothelial layer, but before theaddition of the second endothelial layer. Fibroblasts incorporatedthemselves in the constructs and took up residence in the matrix thatwas sandwiched by the two layers of endothelium (FIG. 47).

Monocytes are one type of DC precursor; they can also develop intomacrophages. Monocytes were seeded onto the dual endothelial/amnionconstructs, and migration of monocytes across the entire construct wasobserved, although many monocytes stayed relatively close to theoriginal endothelial layer (FIG. 47). This demonstrates that all partsof the matrix are “cell friendly” and accessible for migration. Incontrast to neutrophils, monocytes can migrate across endothelium in theabsence of inflammation, because endothelium constitutively produces thefactors that support their migration.

In another embodiment, a good choice of matrix may be acellular humandermis itself; it is feasible to make such matrices from the splitthickness skin discussed earlier. There are several methods to removethe sheet of epidermis (Dispase or ammonium thiocyanate), and remainingsheets that are of appropriate thickness can be selected to establishthe desired construct in which vascular endothelium is cultured on oneside and epidermis on the other. This design takes into account theincorporation of transmigrating blood cell precursors into Langerhanscells in the epidermis, DCs of the dermal type in the matrix, togetherwith macrophages that take up residence in the matrix.

Example 15 Communication between VS and LTE

The VS can be placed in communication with the artificial lymph node(LTE) of the present invention. Such direct communication can beachieved by inclusion of a flow chamber that permits such communication.For this embodiment, digital printing technology may be desirable.

FIG. 20 illustrates aspects of the present invention. In an embodiment,scaffolding for flow-supported, cannulated, endothelial cell lined tubesare constructed using digital printing technology. Preferably, this willinclude an epithelial layer (IV), but can also be a more simple vascularstructure (III). The red arrow indicates the 3D nature of the constructin which the tubes are fully surrounded by connective tissue matrix. Inan embodiment of the present invention, the construct has lymphatic andvascular tubes intertwined and positioned near dermal papillae, asoccurs naturally in skin.

With this advanced in vitro skin equivalent construct that replicatesthe functional architectural and immunophysiology of natural skin, it ispossible to test compounds in vitro. The VS is an accurate, reliable andreproducible means to replace animal systems for safety testing ofcosmetics, chemicals, lotions, creams, adjuvants, vaccines, drugs,biologics, and other compounds.

For reasons of safety and risk assessment, new chemicals are presentlyevaluated for irritant potential by application to animals andobservation of visible changes, such as erythema (skin redness) andedema (accumulation of serous fluid). Testing for skin irritation inanimals potentially causes them pain and discomfort and the results arenot necessarily predictive of those in humans. In recent years, animaltesting for dermatotoxic effects has come under increasing scrutiny andcriticism from animal-rights activists as being inhumane andunnecessary. Attempts have been made to restrict the marketing ofproducts containing ingredients that have been tested on animals. Theoften conflicting needs to protect worker and consumer safety, complywith regulatory statutes, and reduce animal testing procedures areleading to a significant effort within industry, government, andacademia to develop alternative testing methods for assessing the skincorrosion and irritation hazards of chemicals and product formulationswithout reliance on animal test procedures.

There is a continuing need for standardized, validated in vitro assaysthat provide dependable, predictive safety data for the broad range ofproducts in the cosmetic, personal care, chemical, household products,and pharmaceutical industries. Factors driving the development of invitro models include the frequent lack of correlation between animaldata and the human response, the importance of human correlation testdata using 3D tissue engineered constructs, increased sensitivity,better control conditions, better experimental flexibility, easierdiagnostics, and the significant expense and time required for in vivoanimal studies.

A major advantage of an in vitro model is that it boasts a rapidturn-around time for data analysis and decision-making via a clearerinterpretation of sophisticated multi-endpoint data. The most prominentquestion guiding dermato-toxicologist is whether skin equivalentscomposed of keratinocytes and fibroblasts are sufficient to evaluate theirritant potential of substances. Certainly, a significant differencebetween in vitro equivalents and natural skin is the composition of thecells and the absence of communication with other tissues.

For evaluation of visible symptoms following irritation, the interactionof blood vessel endothelium, inflammatory cells, and nerves is necessaryand heretofore has been largely ignored. In the present invention, anadvantage is that the VS comprises blood and lymphatic endothelia aswell as various immune cells (monocytes, dendritic cells, mast cells,macrophages, neutrophils, fibroblasts), cell types important forevaluating inflammatory responses to irritants, scaffold materials, andthe assemblage of such a construct. These additions are important toachieve a valid physiological response to properly assess the cosmetic,chemical, drug, biologic, vaccine or adjuvant in question.

An embodiment of the present invention comprises a tissue engineeredscaffold to integrate vascular tubes into constructs lined with vascularand lymphatic endothelium, with and without epidermal epithelium. Thisrequires identifying conditions that permit growth of the desired celltypes in a cannulated scaffold at a known blood flow rate throughpost-capillary venules. With this design, the result is a more realisticvaccination site. For example, with a continuous flow loop of leukocytesthrough the vascular compartment, administration of an inflammatorystimulus to the connective tissue space would allow all recruitment ofcells to occur with their normal kinetics and normal endothelial cues,such that the endogenous environment would orchestrate the entireresponse to the administered vaccine. Cells could be collected from thelymphatic cannula for analysis, or this cannula could be established tocommunicate with the artificial lymph node

In a tissue engineering approach to forming a VS tissue, havingdetermined desired cell types and scaffold characteristics, in oneembodiment, a digital printing system is used to form the scaffold andto seed cells into a configuration amenable to the final AIS bioreactorformat. Deposition conditions include:

1. BAT handling of cells,

2. optimal scaffold pore size and framework dimensions,

3. scaffold characteristics for each tissue type,

4. interfacing/connecting scaffolds for various tissue types andfluid/nutrient paths,

5. materials to prevent cell adherence to non desired bioreactorsurfaces including adherence of cells to scaffold areas devised forother tissue types,

6. cell seeding density for each tissue type,

7. order of cell seeding (simultaneously or staged; and how to achievein final bioreactor configuration), and

8. integration and digital printing of methods into final bioreactorconfiguration.

Difficult engineering challenges in making engineered human tissuesaccording to the present invention include the delivery of nutrients anduniform seeding of cells throughout the scaffold or growing tissueconstruct. In a preferred embodiment, the digital printing BioAssemblyTool (BAT) system is used to create 3D interwoven structures ofnutrient, oxygen and tissue paths. The BAT prints cells into theappropriate path and the materials used to make each path are conduciveto growth for a particular type of cell, but inhibitory to others. Forexample, only endothelial cells would attach to and line oxygen deliverychannels. Only stromal and parenchymal cells would anchor in the tissueareas. Adjacent areas will likely have to be inhibitory to undesiredcell types, as it is our experience that if an engineered tissue is putin contact with a 2D surface, cells migrate out of the tissue and formmonolayers on the 2D surface. Because of this, in the present invention,3D cultures are preferably grown suspended in medium, away from any 2Dbioreactor surfaces. In a preferred embodiment, the BAT can be used tocreate this structure.

If an engineered tissue adequately mimics the in vivo setting, thetissues will respond as they do in vivo. Hence, it is possible that ifan engineered skin equivalent is injected with an antigen and put incontact with a fluid stream containing monocytes, the skin equivalentitself may create the chemokines necessary to attract monocytes.

Immature DCs are recruited to sites of inflammation in peripheraltissues following pathogen invasion; this is to directing cells to theVS (skin equivalent) in the in vitro immune system of the presentinvention. Internalization of foreign antigens can subsequently triggertheir maturation and migration from peripheral tissues to lymphoidorgans. Chemokine responsiveness and chemokine receptor expression areimportant components of the DC recruitment process to sites ofinflammation and migration to lymphoid organs Immature DCs may expresschemokine receptors including CCR1, CCR2, CCR5, CCR6, and CXCR1.14, 15.They can thus be chemoattracted to areas of inflammation primarily byMIP-3β, but also in response to RANTES (regulated on activation, normalT cell-expressed and secreted)/CCL5 and MIP-1α (macrophage inflammatoryprotein-1α)/ CCL3.16. Following antigen acquisition and processing, DCsmigrate to T cell rich areas within lymphoid organs via blood or lymph,simultaneously undergoing maturation and modulation of chemokine andchemokine receptor expression profiles. A change in expression levels ofthe chemokine receptors CCR6 and CCR7 contributes to the functionalshifts observed during DC maturation.

To orchestrate all of these migratory routes to the VS (monocytes), outof the VS (mature DCs), and into the LTE (mature DCs, T and B cells), itis an important feature of the present invention to incorporatechemotaxis into the in vitro immune system. An importantmaterials/device design feature is the incorporation of the messages,soluble and insoluble molecules that promote cellular attraction.Further, development of a complete LTE requires signal delivery to T andB cells as well, to provide cues to organize these cells on the LTEscaffold.

Example 16 The Rapid Chemokine Testing System

The BioAssembly Tool (BAT) has been modified for use in observations ofthe lateral motility of cells in experiments on chemotaxis. Thedescribed setup enables rapid screening of chemokines and cell matrices(typically, one experiment can take about half to one hour).

Three chemokines, fMLP-FITC; MIP-β (macrophage inflammatory protein-β),and MIP-3β, were tested with human monocytes. It was found that thesaturation level for fMLP FITC(N-formyl-methionyl-leucyl-phenylalanine-fluorescein isothiocyanate)(˜40 nM) was close to that known for fMLP(N-formyl-methionyl-leucyl-phenylalanine) (˜30 nM). MIP-3β was foundineffective (as expected for immature monocytes); MIP-3α was foundeffective, despite negative information and expectations. It was alsofound that fibrin glue effectively arrested cell motility even at lowconcentration (˜1 mg/ml), whereas collagen favored motility. Thus,fibrin matrices should be used preferably in cases when cell retentionis required, and collagen should be used to make cell-permeablebarriers. This latter observation may be important when considering theuse of ETCs for wound healing procedures. A schematic picture of therapid chemokine testing system is shown in FIG. 21. This system can beused to further refine and optimize chemokine doses using variousmicroparticle strategies and formulations, temporal releasecharacteristics, and scaffold characteristics (e.g., geometry, porosity,material) on cell migration.

The rapid chemokine testing system can be used to examine saturation ofcell receptors by chemokines. When a chemokine reaches its saturatingconcentration, cells become insensitive to the signal and stop theirlocomotion. In most cases, chemotaxis occurs through gradients ofchemokines. The steeper the gradient, the more effective the attraction;in the same time, the shorter the distance that can be covered by thegradient before saturation is achieved.

Example 17 Cell Maturation in LTE

This example relates to the lymphoid tissue equivalent (LTE) which isalso referred to as the artificial lymphoid tissue. LTE is the commandcenter of the artificial (ex vivo) immune system that contains the naiveand/or memory T and B lymphocytes. T and B cells play key roles inadaptive immunity by destroying infected cells, producing antibodiesthat opsonize pathogens, and secreting cytokines that induce effectorfunctions in other immune cells. Activation of naive lymphocytes occurswithin secondary lymphoid tissues (including lymph nodes, Peyer'spatches, spleen). T cells are activated by antigenic peptides presentedto them in the cleft of class I and class II MHC molecules by dendriticcells emigrating from the periphery to the lymph nodes, while B cellsare activated by direct binding of foreign molecules with their antigenreceptors and subsequent interactions with activated T cells.

T cells, B cells, and DCs in lymph nodes are found in two anatomicallydistinct regions (FIG. 22). These are the paracortex or T zone (home toT cells and dendritic cells) and the follicles (home to B cells andassociated supporting cells, including follicular dendritic cells).

During resting homeostasis, T and B cells continuously recirculatethrough the blood between secondary lymphoid organs. T and B lymphocytesenter the lymph nodes from the blood via specialized vessels known ashigh endothelial venules (HEVs) in the paracortex, and are directedtoward the T zone or follicles by specific chemokines produced in eachzone. T and B cells typically reside within a given lymph node for 24-48hrs, and if activation signals are not encountered, they move on tocontinue their recirculation via the blood to other secondary lymphoidorgans.

On initiation of an immune response, these cells leave their respectivezones and follow a program of cell-cell interactions in an orchestratedfashion within the lymph node. These steps in the generation of adaptiveimmune responses are discussed below.

In an embodiments of the present invention, synthetic and/or natural ECMmaterials may be used to fabricate matrices for the LTE to achieve 3Dstructures that provide a physical structure mimicking the lymph node's“open” reticular network, containing lymphocytes, as well as biochemicalcues (e.g., adhesion motifs, chemokine gradients) expected bylymphocytes in secondary lymphoid tissue. In addition, hybrid approachescan be used that combine the controlled microstructure design ofsynthetic approaches with the more native materials of natural ECM.Example LTE structures comprising segregated T and B zones can befabricated with overall structures mimicking the physical arrangementshown in FIGS. 23A-23I. Briefly, FIGS. 23A and 23B is an image oftoluidine blue-stained HUVEC cells on a collagen cushion, showingcharacteristic cell packing. From the time of seeding the HUVEC cells ona collagen cushion, it typically takes about 5 days from confluency tooccur and for the cells to take on the normal vascular endothelialmorphological characteristics. FIG. 23C shows a high density of newlyapplied peripheral blood mononuclear cells (PBMCs) on the layer ofHUVEC. FIG. 23D shows a focal plane below the HUVEC cells, within thecollagen matrix, 45 minutes after the application of PBMCs. Cells infocus are within the collagen and are easily distinguished between HUVECand surface PBMCs. In FIG. 23E, CMFDA labeling was done to show cellviability and position of live cells within the collagen cushion. FIG.23F shows transmigration of PBMCs into collagen cushions without or withthe presence of Zymosan. Phase contrast, and CMFDA labeling was done todetermine cell placement within the cushion. Z-stack images were takenthrough the entire cushion to determine the numbers of cells within thecushion and those that had undergone transmigration. Data analysisshowed increased numbers of transmigrated cells remained in the cushionin the presence of Zymosan as compared to cushions with no Zymosan(FIGS. 23H and 23G). Transmigrate cells in the presence of Zymosan didnot penetrate as deeply, because of the stimulatory nature of Zymosan.

Characterization of the HUVEC endothelial cells in the collagen cushionis paramount to showing a confluent layer, and appropriate morphologicalcharacteristics which will allow the highest number of PBMCs migratinginto the collagen cushion. As shown in FIG. 58, the HUVEC cells maintaina confluent monolayer with typical morphologic characteristics after 10days of culture (panel B: These morphological characteristics areimportant to maintain prior to PBMC application.

The role of lymphoid tissue stromal cells in supporting T and B cellfunctions in these LTE matrices can be assessed to further determine howlymph nodes form in vivo and to identify key factors controlling lymphnode self-organization.

Primary immune responses are initiated by dendritic cells presentingforeign peptides in the cleft of major histocompatibility complex (MHC)molecules to the T cell receptor (TCR) of antigen-specific, naive Tcells (Banchereau & Steinman, Nature 392:245-252 (1998); Banchereau, etal. Annu. Rev. Immunol. 18:767-811 (2000)). On contact with theircognate antigen presented by DCs, T cells remain in lymph nodes for 2-4days, undergoing differentiation/clonal expansion and providing help toantigen-specific B cells, before exiting the lymph nodes to carry outeffector functions in the periphery (Butcher, et al., Adv. Immunol.72:209-253 (1999); Sprent et al., Cell. Immunol. 2:171-181 (1971)).

Naive T cell activation occurs in the T zone of lymph nodes (FIG. 24); Tcells search for antigen-bearing DCs that have migrated to the T zonevia blood or lymph from sites of infection in the peripheral tissues(Garside, et al., Science, 281:96-99 (1998); Jenkins, et al., Annu. Rev.Immunol. 19, 23-45 (2001); Kaldjian, et al., Int. Immunol. 13, 1243-1253(2001)).

Unlike the fine collagen fibril mesh of peripheral connective tissues,the extracellular matrix of the T zone is organized in an open, web likesystem of collagen fibers, known as the reticular network (Kaldjian, etal., Int. Immunol. 13:1243-1253 (2001)). These thick (about 0.5 to about5 μm diameter (Gretz, et al., J. Immunol. 157:495-499 (1996)) fibers arespaced about 20 to about 30 μm apart (FIG. 24); the contrast between thestructure of the reticular network and that of a typical collagen gel isillustrated in FIG. 24. The reticular fibers consist primarily ofcollagen I, collagen III, and fibronectin (Kaldjian, et al., Int.Immunol. 13:1243-1253 (2001); Gretz, et al., Supra (1996)) and supportthe attachment of a layer of stromal cells, known as fibroblasticreticular cells (FRCs) via β1-integrins (van den Berg, et al., Am. J.Pathol. 143:1098-1110 (1993); Gretz, et al., J. Exp. Med. 192:1425-1440(2000)). FRCs ensheath reticular fibers and join one another with tightjunctional complexes (Stuart & Davidson, J. Pathol. 103: 41-47 (1971))to form a “living substrate” for T cell and dendritic cell migration(Crivellato & Mallardi, J. Anat. 190:85-92 (1997)).

The T zone is also compartmentalized from bulk flow of lymph or blood,which may preserve a particular controlled microenvironment in the Tzone during immune responses (Gretz, et al., J. Exp. Med. 192:1425-1440(2000)). T cell activation thus occurs within the stromal celllatticework under conditions where exogenous factors are minimized andconversely, factors secreted by dendritic cells and stromal FRCs mayhave maximum potency. Once CD4+ T cells are activated by DCs, they maymigrate to the periphery of the follicles to provide ‘help’ to activatedB cells for proliferation and antibody isotype switching (Garside, etal., Science 281:96-99 (1998)).

While it remains unclear whether the unique microenvironment of the Tzone is absolutely required for naive T cell activation, several linesof evidence point to its importance in mounting efficient and effectiveresponses to pathogens. In mice lacking the chemokine receptor CCR7(CCR7−/−), T cells and DCs do not meet in the T zone and these mice areunable to mount immune responses (Forster, et al., Cell 99:23-33(1999)). Mice bearing a mutation (plt/plt) causing disruption of T zonearchitecture show a high susceptibility to some (though not all)viruses, delayed T cell responses, and aberrant T cellexpansion/survival (Gunn, et al., J. Exp. Med. 189:451-460 (1999); Junt,et al., J. Immunol. 168:6032-6040 (2002); Mori, et al., J. Exp. Med.193:207-218 (2001)). In vitro studies of T cells interacting with DCs in3D collagen gels have shown very different dynamics for the duration andnumber of T cell DC contacts occurring, compared to simple liquid phasecocultures (Gunzer, et al., Immunity 13:323-332 (2000)), and recent invivo studies imaging T cell DC interactions in intact lymph nodesconfirm that these cells behave very differently in their native 3Dmicroenvironment compared to 2D cultures (Miller, et al., Science296:1869-1873 (2002); Stoll, et al., Science 296:1873-1876 (2002)).

Example 18 Activation of B cells

The current model of B cell activation in primary immune responses isillustrated in FIG. 27 (from Baumgarth, Immunol. Rev. 176:171-180(2000)). B cells in follicles first bind soluble antigen carried to thelymph node by the lymphatics, which triggers their migration to the edgeof follicles where they meet activated CD4+ helper T cells. Theactivated CD4+ cells then provide ‘help’ in the form of CD40-CD40 ligandinteractions and cytokines that promote B cell proliferation and isotypeswitching.

This model is in accordance with many studies showing that stronghumoral immune responses to most antigens require the presence ofactivated CD4+ T cells (Fulcher & Basten, Int. Rev. Immunol. 15:33-52(1997); Parker, Annu. Rev. Immunol. 11:331-360 (1993); Goodnow, et al.,Adv. Immunol. 59:279-368 (1995)). This pattern of B cell trafficking hasbeen visualized in lymph nodes of mice (Garside, et al., Science281:96-99 (1998)).

Following interactions with T cells, some activated B cells proceed togerminal centers, specialized regions of the follicles which developduring immune responses. Within follicles, B cells proliferate andundergo somatic hypermutation, a process designed to geneticallymanipulate the antibody specificity of activated cells to find highaffinity mutants that can more effectively eliminate pathogens.

Specialized dendritic cells (follicular dendritic cells) capture antigenantibody complement complexes and present them to the antibodyrearranging B cells in the germinal centers; those B cells that develophigher affinity antibodies (expressed in membrane bound form on theirsurface) are given survival and proliferation signals, while B cellswhose antibody chains become unable to recognize the antigen apoptose(Kosco Vilbois, Nat. Rev. Immunol. 3:764-769 (2003)). The result is arapidly expanded population of isotype switched, high affinity antibodyproducing B cells, which finally either differentiate into long livedplasma cells or memory B cells.

Two points are particularly relevant for the development of asimplified, functional model of the immune system in vitro, such as theAIS of the present invention. First is the important nature of T cellhelp in producing B cell proliferation and isotype switching. The secondis the lack of clarity as to whether germinal center reactions areabsolutely required for isotype switching, affinity maturation, andmemory cell development.

In addition to effects on cellular states within the lymph node, it islikely that a 3D matrix is important to allow T cell DC interactions andthe T and B cell migration that facilitates delivery of help for B cellactivation and antibody production. Many animal studies have providedevidence that T cell help is important for strong antibody responses.Thus, a lymphoid tissue equivalent must support T cell-B cellinteractions. In the present invention, the ‘matching up’ of rareantigen specific T and B cells is achieved via a LTE structure thatallows the natural “self assembly” process that brings these cellpopulations together by autonomous migration in the lymph node.

In addition to effects of the 3D supporting matrix, cellularinteractions are important in the lymph node. This likely includesinteractions between lymphocytes and stromal cells. Recent studies haveshown that survival and function of B cells is enhanced in vitro whenthey are cocultured with secondary lymphoid tissue stromal cells(Skibinski, et al., Eur. J. Immunol. 28:3940-3948 (1998)). Lymphocyteswithin the lymph node also have a complex interdependence; for example,dendritic cells secrete factors that promote antibody production and Bcell survival/proliferation (Dubois, et al., J. Exp. Med. 185:941-951(1997)). All of the cell types that can be part of the LTE (including Tcells, B cells, DCs, lymphoid stromal cells) have the potential tointeract and influence one another.

Example 19 LTE Structure and Germinal Centers

The LTE serves as an important locus for activation of naive T and Bcells. The present invention includes, in the design of the LTE,multiple approaches for fabrication of a model of the lymph nodeextracellular matrix and providing various microenvironemental cues(such as chemokines, cytokines, cells (e.g., fibroblastic reticularcells)). Specific design considerations for the LTE include:

1. using synthetic and/or natural lymphoid ECM derived hydrogel matricesas models of the reticular network ‘scaffold’ of the lymph node.

2. The role of matrix composition and presence of supporting lymphoidstromal cells on T cell activation and DC survival/function within theLTE.

3. Fabrication of LTE structures comprising both T and B zones. Thesewill be assembled using several complementary strategies.

-   -   a. Direct physical assembly of segregated T and B cell areas.    -   b. Self organization and maintenance of T and B cell areas via        creation of engineered local chemokine sources within distinct        locations with the matrix.

The following description sets out in detail the experimental rationaleand approach for each of these features of the present invention.

Clearly, the dynamic multi cellular interactions occurring during animmune response in the lymph nodes or other secondary lymphoid tissuesrepresent a complexity significantly above anything attempted in an invitro model of tissue or organ function to date, except in whole, exvivo organ cultures. To manage the complexity of this problem, thepresent invention is limited to a model of aspects of lymph nodephysiology that are, relatively, quite well understood and likelyimportant for basic functions of the lymph node.

Germinal centers represent one of the most complex and dynamic tissuemicroenvironment in the body; their function is as yet poorlyunderstood. Affinity maturation occurring within germinal centers is anextremely complex process that is not well understood; both the stromalcells and follicular dendritic cells that are involved and localmicroenvironmental factors (cytokines, chemokines) remain poorly defined(Kosco Vilbois, Nat. Rev. Immunol. 3:764-769 (2003); Cyster, et al.,Immunol. Rev. 176:181-193 (2000)).

The LTE of the present invention thus lacks any ‘engineered’ germinalcenters or germinal center precursors. This choice is based on thefollowing experimental observations. First, B cells from mutant mice(lymphotoxin-α- or tumor necrosis factor-α-deficient) lacking germinalcenters are still able to undergo antibody isotype class switching andsomatic mutation, and further produce high-affinity antibodies inresponse to antigen, suggesting that germinal centers are not absolutelyrequired for affinity maturation (Matsumoto, et al., Nature 382:462-466(1996); Pasparakis, et al., J. Exp. Med. 184:1397-1411 (1996)). Second,functional B cell memory (defined by rapid production of high titer IgG1in response to antigen re-challenge) has been found to be intact in micelacking Bc16, which also do not form germinal centers (Kosco-Vilbois,Supra (2003); Toyama, et al., Immunity 17:329-339 (2002)). Finally, invitro studies have shown that B cells cultured with activated T cells(or surrogate cells providing CD40L signals) are capable of promoting apartial germinal center phenotype, isotype switching, and somaticmutations (Galibert, et al., (1996) J. Exp. Med. 183:77-85; Razanajaona,et al., J. Immunol. 159:3347-3353 (199&)).

A second simplification in the LTE of the present invention is the lackof programmed naive T cell/B cell recirculation; cells loaded into theLTE are not exposed to structures mimicking high endothelial venules(HEV) that might promote exit from the lymph node during homeostasis. Invivo, T cells depart a given lymph node to recirculate among the varioussecondary lymphoid tissues via the blood every 24-48 hours. In analternative embodiment of the present invention, an in vitro model ofHEV monolayers atop the LTE structure is contemplated (FIG. 26). In amurine system, high endothelial cells have been isolated (Phillips &Ager, Eur. J. Immunol. 32:837-847 (2002); Rot, J. Immunol. Methods273:63-71 (2003)).

In one embodiment, the LTE comprises a synthetic hydrogel ‘inverse opal’matrix. Ordered macroporous hydrogels are prepared by pouring apoly(ethylene glycol) (PEG) dimethacrylate and PEG peptide PEG blockcopolymer solution over an ordered colloidal crystal of poly(methylmethacrylate) latex microspheres (monodisperse with diameters of about5-150 μm) and UV polymerizing the gel (FIG. 27A). Microspheres are thenleached out by brief treatment with acetic acid followed by extensivewashing of the gel with phosphate buffered saline (PBS). Shown in FIG.27B) is an example of the ordered honeycomb like structures obtained bythis method; the sequence of four smaller images is a 3D reconstructionof one “cell” of the scaffold in rotation, showing the “side ports”connecting this pore of the scaffold to its neighbors in the x-y plane.As illustrated in FIG. 60, scaffolds of arbitrary shape and dimensionscan be synthesized, of both macroscopic or microscopic dimensions.

Use of peptide containing crosslinkers in the gel allows tailored ECMmimicking peptides, or complete ECM proteins as desired, to be includedin the gel (FIG. 27C) (Irvine, et al., Biomacromol. 2:85-94 (2001); West& Hubbell, Macromolecules 32:241-244 (1999)). Adhesion sequences canalso be included in the hydrogel to promote cell attachment andmigration in the structure. In addition, the design incorporates enzymesensitive cross linkers that allow cells to remodel the structure usingnative pathways (e.g., collagenase). With this system, well defined ECMmimetic structures with tailored pore size, mechanical properties, andbiochemical composition are designed in.

To complement the above approach to fabricating a suitable matrix forthe LTE, the present invention also includes a approach to fabricatinghybrid synthetic/natural ECM structures, in which one can apply thecolloidal crystal templating method to fabricating natural ECM scaffoldswith a defined architecture. In these experiments, the hydrogelprepolymer solution in the templating step (FIG. 27A) can be replacedwith ECM gel in its liquid form. The gel was subsequently solidified at37° C. in the presence of the templating spheres, cross link the ECM inplace covalently, and dissolve out the templating spheres with aceticacid. This approach combines the native biochemical structure of the ECMgel with the defined microstructure of the synthetic inverse opalstructures.

Example 20 Microbeads Fabricated from Lymphoid Extracellular Matrix

Microbeads were fabricated from porcine lymphoid extracellular matrixprepared using a protocol provided by Dr. Stephen Badylak, University ofPittsburgh.

A suspension containing ˜10 mg/ml lymph node (LN) ECM microfragments in2 mg/ml Protasan, pH 3.5, was sprayed over the surface of liquidnitrogen in a laminar, drop-by-drop mode, making droplets of about 1.5mm in size. The frozen beads were then freeze dried overnight, incubatedin 10% tripolyphosphate (TPP), pH 6.0, for 1 hour thereafter, thenwashed three times with deionized water over a 100 μm cell strainer, andwere then freeze-dried again (FIG. 28).

Example 21 Loading LTE with Chemokine and Lymphocytes

In another embodiment of the present invention, the LTE comprises amicrocarrier loaded with a chemokine and lymphocytes. Another embodimentof the invention relates to a method of constructing the LTE; saidmethod comprises (1) providing matrix; (2) loading said matrix with achemokine; and (3) cultivating lymphocytes with said matrix.

In order to attach B and T cells to microcarriers, B and T cellfractions were negatively selected from peripheral blood lymphocytesusing a magnetic bead-based separation protocol. Cell suspended in PBSwere deposited onto various microcarriers and incubated for 1 hr. Themicrocarriers were then washed in PBS four times, and attached cellswere revealed on the surface of carriers by the green fluorescence ofthe internalized CFSE stain (FIG. 29(A),(B),(C)).

The microcarriers can be saturated with chemokines. In one embodiment,CXCL-13 (BCA-1; BLC) and CCL-21 (SLC; Exodus-2) were chosen as basic Band T cell specific chemokines, respectively.

Many chemokines are strongly basic proteins; that is, they bear aminogroups which are positively charged at neutral pH (Proudfoot, et al., J.Biol. Chem. 276:10620-10626 (2001)). On the other hand, manymicrocarriers also contain positively charged groups (e.g., diethylaminoethyl fragments in Amersham products; amino groups inchitosan/Protasan). Consequently, to provide for proper attachment ofchemokines, charge modification of the surface of the carriers isimportant.

As an example, this could be accomplished using polyanionic mediatorsthat attach to positively charged microbeads and overcompensate thecharge, according to the known mechanism of layer-by-layer (LBL)supramolecular assemblies (Kotov, NanoStructured Materials 12:789-796(1999)), thus providing for subsequent reversible electrostaticattachment of positively charged chemokines.

Heparin, a natural component of extracellular matrix, was chosen as amediator. Heparin contains multiple pentasaccharide units bearingsulfate and carboxylic groups; the average charge per unit ratio is 2.3(Sasisekharan & Venkataraman, Current Opinion in Chemical Biology4:626-631 (2000)) (FIG. 30).

In an embodiment, cytopore-1 microcarriers (Amersham), 10 mg total, weresoaked overnight in 1 ml PBS buffer containing 10 mg porcine heparin(Sigma-Aldrich); another portion of Cytopore-1 was soaked in the buffercontaining no heparin. The samples were washed afterwards 7 times withcopious volumes of deionized water and 1 time in PBS containing 0.1%bovine serum albumin (BSA), and transferred into glass tubes containing2 ml PBS/BSA plus ˜200 ng/ml BLC chemokine. Upon incubation overnight,the samples were analyzed for BLC remaining in solution and absorbed onCytopore microcarriers using a one-step Quantikine ELISA kit (R&DSystems) (FIG. 31).

Example 22 Tissue and Matrix Effects on T Cell Activation in the LTE

Both the extracellular matrix and stromal cells of lymph nodes likelyplay significant roles in T cell, B cell, and dendritic cell function inthe secondary lymphoid organs. From the standpoint of the cellularmakeup of lymph nodes, stromal cells of the T zone are likely to play asignificant role in T cell activation, via production of cytokinesand/or chemokines, as well as the expression of receptors that support Tcell and DC migration through the T zone.

Derivation of T zone stromal cells. The design of the LTE in the presentinvention includes the engrafting of T zone fibroblastic reticular cells(FRCs, stromal cells of the T zone) on the hydrogel scaffolds. As anexample, stromal cells were isolated from lymph nodes of C57BL/6 mice ina manner similar to previous reports (Bogdan, et al., J. Exp. Med.191:2121-2130 (2000); Castro, et al., Eur. J. Cell Biol. 74:321-328(1997); Skibinski, et al., Immunology 102:506-514 (2001); LeBedis, etal., Int. J. Cancer 100:2-8 (2002)) to test the response of these cellsto our synthetic materials. The stromal cells thus obtained had acharacteristic fibroblast-like morphology (FIG. 32A). Stromal cells oflymph nodes express high levels of CD44 and VCAM 1 (Skibinski, et al.,Eur. J. Immunol. 28:3940-3948 (1998); Ruco, et al., Am. J. Pathol.140:1337-1344 (1992)); flow cytometry analysis of FRCs stained with antiCD44 and anti VCAM 1 confirmed expression of these molecules by thestromal cell lines (FIG. 32B). Synthetic poly(ethylene glycol)-basedhydrogels presenting RGD adhesion peptides supported the attachment,spreading, and growth of FRCs over several days in culture (FIG. 32C).These cells also grew well on fibronectin, collagen IV, and collagenI-coated surfaces, but not on laminin (data not shown).

Example 23 Assembly of T and B Cell Areas

To provide the lymph node function of T cell help for B cell antibodyproduction, distinct T and B cell areas within the LTE of the presentinvention are fabricated by the combined action of digital printing(directly assembling T cells and B cells within distinct zones) and/orcontrolled release technologies (e.g., using microspheres releasing Tand B cell attractants to maintain T and B cell areas, respectively).Together with the materials used to fabricate the matrix, this allowsspatial and temporal control over the model lymphoid microenvironment,to properly tune cell migration and cell-cell interactions: bothadhesion ligand and chemokine type, spatial location, density, andconcentration with time can be varied to optimize lymphocyte functions.While full control over all of these variables provides the fullestmimic of the in vivo environment, as illustrated in the data given abovefor model LTE structures, some limited subset of adhesion molecule,chemokines, and other soluble factor information in the LTE can achievesignificant functionality within these in vitro constructs.

Digital printing of heterogeneous LTE zones. The Bio-Assembly Tool (BAT)allows the deposition of viscous liquids, including live cellsuspensions with micrometer scale precision, using computer control.Sequential depositions allow 3D structures to be built, as illustratedby the mock lymph node gel structure (FIG. 33A). The digital printingability of the BAT is used to co-deposit lymphocytes with or withoutcontrolled-release microspheres into designated ‘zones’ within LTEscaffolds, comprising, e.g., the synthetic and/or natural ECM matrixexamples described above.

Example 24 Maintenance of T and B Cell Areas Using Chemokines

T and B lymphocytes are highly motile, and one might expect that if themicroenvironment of the LTE matrix supports migration, the T cell and Bcell areas created by direct printing into the LTE will not remainwell-defined over time in culture. In vivo, these zones are believed tobe maintained by the action of local chemokine gradients that locallyattract T and B cells to their respective compartments within secondarylymphoid tissues (Cyster, et al., Immunol. Rev. 176:181-193 (2000);Cyster, J. Exp. Med. 189:447-450 (1999)) (FIG. 34A). To provide adefined simulation of these conditions in the LTE, an embodiment of theLTE of the present invention includes the co-deposition of cells withchemokine releasing microspheres, designed to provide a local center ofgravity for each cell type in their local zone (FIG. 34B).

Distinct chemokines serve to organize the T cell and B cell areas oflymph nodes; CXCL13 (BLC) is known to be a key factor localizing B cellsin lymph node follicles (Cyster, et al., Immunol. Rev. 176:181-193(2000); Ansel, et al., Nature 406, 309-314 (2000)), while CCL19 (MIP-3β)and CCL21 (SLC) draw T cells and dendritic cells to the T zone (Cyster,J. Exp. Med. 189:447-450 (1999); Mebius, Nat. Rev. Immunol. 3:292-303(2003)). In an embodiment of the present invention, lymph nodefibroblastic reticular cells can be included in the T zone of the LTE toobtain a “native” source of chemokines to self organize the T zone ofthe scaffold. In another embodiment, the LTE includes controlled releasemicrospheres to obtain defined chemoattractant depots within the LTE.For example, the use of degradable poly(lactide-co-glycolide) (PLGA)microspheres to chemoattract dendritic cells and monocytes using formylpeptides or the chemokine MIP-3β has been demonstrated. For selforganization of the LTE, similar procedures can be used to encapsulateBLC, MIP-3β, and SLC in microspheres for controlled release within theLTE matrix. As both MIP-3β and SLC are involved in T zone organization,these can be included separately or in combination. One then canco-deposit microspheres with T cells and B cells into LTE scaffolds, andcompare the maintenance of defined T cell and B cell areas within thesestructures over time in these chemokine directed scaffolds, comparedwith matrices that lack such microspheres. In another embodiment, it ispossible to directly encapsulate microspheres within the ‘struts’ of thehydrogel matrix during polymerization. In still another embodiment,FRC-engrafted T cell areas can be prepared to determine whether thesestromal cells can guide T cell localization within scaffolds.

Example 25 In Vitro Tissue Slice Templates

Additional approaches to constructing a functional LTE. The embodimentsabove describe an approach to fabricating a minimal, functional mimic ofmammalian, preferably human, secondary lymphoid tissue. Otherembodiments considered within the scope of the present invention are nowdescribed.

Another embodiment involves ‘templating’ the LTE using native humanstromal cells (FIG. 35), in a manner similar to that reported byresearchers attempting to develop an in vitro artificial thymus(Poznansky, et al., Nat. Biotechnol. 18:729-734 (2000)). Their approachcomprised the following steps:

1. small thymus fragments from mice were cultured on the surface of CellFoam disks (a porous matrix) in 12-well plates and covered in growthmedia for 14 days until a confluent layer of stroma had formedthroughout the matrix.

2. upon reaching confluence, human lymphocyte progenitor cells wereadded into the co-culture.

3. during co-culture for 4 to 21 days, non-adherent cells wereperiodically harvested and cell surface markers were analyzed todetermine T lymphopoiesis.

Following a similar scheme, in an embodiment of the present invention,LTE matrices could be “templated” with stromal cells derived from lymphnode fragments or lymph node, spleen, or tonsil “slices” to seed theconstruct with native stromal cells and provide a ready microenvironmentfor added T cells, B cells, and DCs. Such cocultures can be maintainedin vitro using standard organ culture methods during the templatingstep, and the templated LTE can subsequently be loaded into the AISbioreactor for continued maintenance. This approach not only provides analternative for generating a correct lymphoid microenvironment, but alsoa complementary in vitro approach for analysis of lymph node formationand organizing principles.

Example 26 Sources of Cells to Populate the Artificial Immune System

To populate the in vitro immune system, a large number of cells,including monocytes, T and B cells, endothelial cells, fibroblasts,keratinocytes, stromal cells, as well as neutrophils, mast cells, andother immune cells may be needed. In one embodiment, a ready source ofimmune cells is peripheral blood (PBMC), which will provide many of thecells needed for the vaccination site and for the LTE. In anotherembodiment, skin related cells from human samples (described in thevaccination site section) can be used. In still another embodiment, itis possible to use the HuScid mouse as a source of hematopoietic cellsthat are generated from CD34+ stem cells in vivo. Additionally, asmethodologies for generating all these cell types from embryonic stem(ES) cells are developed, the present invention is considered to includeusing completely matched cells derived from one ES cell strain/line.

Example 27 Bioreactor Design and Construction: Integration of the AISComponents

Drawing an analogy with high throughput drug screening technology, anAIS suitable for rapid vaccine or chemical screening will requiremultiple, low-cost, disposable bioreactors, designed for single-use.Each bioreactor will be challenged with a different antigen and, uponactivation of the immune response, harvested for antibodies, B cells,and T cells. Microfluidic bioreactors are preferred for achieving thisgoal and provide the additional advantage of requiring fewer scarcecells for seeding tissue constructs.

As illustrated in FIG. 36, in an embodiment, the AIS bioreactor can befabricated as a two-compartment microscope slide with a transparentpolymer sheet or glass coverslip for microscopic examination. In apreferred embodiment, the physical dimensions of each immune bioreactormeasure of the order of about 7.5 cm long and about 2.5 cm wide, with anoverall thickness of about 2 mm or less. The first chamber contains theVS and LTE membranes that can be grown as modular units and laterinserted into the lower structural layer or as a fully integrated systemfrom the start. The second chamber contains the LTE, comprising T and Bcell populations. If required, additional LTE constructs can be added toenable lymphoid organ trafficking or trafficking to other tissues.Syringe tube ports located on the upper layer permit injection offactors and/or cells at strategic positions along the vascular pathwaysand within ETCs. FIG. 48 shows a plan view of an example integratedbioreactor that shows micromachined blood vascular and lymphaticpathways with high contact area beneath the VS and LTE ETCs.

To promote interaction between cells migrating along the blood vascularand lymphatic pathways and in the VS and LTE tissue constructs, thecontact spacing between each tissue membrane can be adjusted by using,e.g., machined inserts or thin laminates that have small, integratedmicrochannels. Suitable construction materials include biologicallycompatible polymers, such as polycarbonate, polyethylene, or acrylic. Alaminate-based insert is as shown in the example (FIG. 37), where as alarger milled tubular design is incorporated in to the designillustrated in FIG. 36. In a sense, these designs mimic a thin venulepathway that supports lymphocyte migration from peripheral blood intosecondary lymphoid organs.

Nutrient-rich media is pumped from an external media reservoir throughthe channels, flowing tangentially past the VS and LTE constructs, andback to the reservoir. Nutrient and waste product transport between therecirculating media and the tissue constructs occurs through bothdiffusional and convective (Starling flow) processes.

In contrast to other nutrients, oxygen is only sparingly soluble in cellculture media. Consequently, high perfusion rates may be required tosustain a sufficient oxygen supply and to avoid developing necroticzones. Should required perfusion rates exceed physical capabilities(e.g., unusually high pressure drops can compromise the integrity ofbioreactor seals) or generate excessive fluid shear, in alternativeembodiments, the oxygen tension in the media may be increased by, forexample, using an O2 microexchanger in-line with the circulating bloodmedia. By circulating the blood media over gas permeable polymers,exposed to high oxygen concentrations on the opposite side, the O2environment can be adjusted to compensate for any O2 consumption andloss. Monitoring and making adjustments to the O2 concentration in thebioreactor can be accomplished using commercially available non contactfluorescent probes to provide feedback to an oxygen air supply. Creatinga high concentration gradient between the gaseous oxygen at the polymerinterface and the tissue construct, can facilitate diffusional transportand culturing of thicker constructs. An example of an assembledconstruct with transparent covers for optical inspection/fluorescentimaging is shown in FIG. 38.

Example 28 Fabrication and Assembly of Layered AIS

Fabrication of such microfluidic bioreactors may require ultra shortpulse machining trials with the biocompatible materials to determineoptimum processing conditions (such as laser fluence and translationspeed). The design of the present invention is sufficiently flexible toallow laser machining of a layered device (e.g., gas permeable polymertop layer, BAT deposited middle layer, and PDMS bottom layer) foradditions of vias or ports after the device has been assembled.

FIG. 49 shows cross sectional views of direct deposition in anembodiment of an AIS device. Various biomaterial structures can beincorporated as constituents of the artificial immune system (e.g., bioconcrete, inverse hydrogel opal, colloidal particles, ECM gels, collagengels, microcarriers). For example, a polymeric mesh rebar can bedeposited layer by layer directly in the recessions of the VS and LTEareas. In such a design, it is preferred to have the lower plate of theAIS unit made of polyacrylate, polystyrene, or another transparentplastic sensitive to DM, to allow the mesh rebar to attach to the plate.In this embodiment, the surface will be micro-patterned using KOH in amanner similar to the ESC scaffolds. Fibrin gel matrix bearing allnecessary nutrients and cytokines will be used to coat the threads ofthe mesh as a thin film, leaving sufficient space for cell accommodationand motion.

As shown in FIGS. 50 and 51, the design of the present invention issufficiently flexible to allow laser machining of a layered device(e.g., gas-permeable polymer top layer, BAT-deposited middle layer, andPDMS bottom layer). FIG. 52 provides a schematic diagram of a perfusedbioreactor system with the associated external pumps for the lymphaticand blood vascular loops and external media reservoirs. The AISbioreactor can be operated in either semi-batch or continuous mode.

In an embodiment of the present invention, integration of membranes inthe bioreactor is achieved by crimping the membranes between thin metal(e.g., stainless steel) rings, as illustrated in FIG. 53. Using such acrimping method, biological membranes can be supported without use ofadhesives and can be pressed into a disk with thickness profile of about400 μm or less.

FIG. 54 shows the fabrication of a 3-layer planar waveguide. FIG. 55shows an example device comprising a perfusion bioreactor, ELISA chipwith integrated optical waveguides, microfluidic backplane to connectand allow swapping of devices and microfluidic connectors for externalpumps and reservoirs.

In addition to machining channels directly, molds can be machined insuitable materials to create a reusable master from which PDMS devicesmay be formed. This will allow a higher volume of devices to befabricated than laser machining in serial. Channel encapsulation methodswill be evaluated to provide a leak-proof construct. The materials thatcomprise the device will likely be damaged at high temperatures, sorobust, low-temperature bonding methods will be needed.

Testing of the devices will require fixtures for mounting and providingexternal connections. Laser machining can also be used to providemanifolds for these test fixtures that would support fast swapping ofdevices without the need to disconnect external pumps or reservoirs.Equipment for measuring pressure, flow resistance and flow rate can alsobe connected to the devices via the manifold. Revisions to optimize thechannel geometries can be made based on this data and performance of theETCs.

The AIS microfluidic bioreactor system can be placed in an incubatorthat maintains constant temperature, humidity, and carbon dioxidecontrol. Phenol red can serve as a colorimetric pH indicator in themedia, so that pH can be monitored, e.g., periodically through visualinspection or photometric determination with logging capabilities. Inanother embodiment, pH can be monitored continuously and precisely inthe external media reservoir with a pH probe and recorder.

Design of the flow channels to control shear forces is important to aidin cell migration through the membrane and to minimize cell stress ordamage. Studies and modeling have shown that by applying shear forces ina controlled range near 2.5 dynes/cm2, lymphocyte migration across anendothelial membrane can be improved (Cinnamon, et al., Nature Immunol.2:515-521(2001)). It has also been shown that elevated shear rates onthe order of 70 dynes/cm2 can damage cells and alter cell function(Moazzam, et al., Proc. Natl. Acad. Sci. USA 94:5338-5343 (1997);Johnson, Biophys. J. 67:1876-1881 (1994); Hochmuth, J. Biomech. Eng.115:515-519 (1993)). Channels dimensions in the bioreactor arepreferably modified to create minimal shear stresses. Preferably, theinlet ports are maximized in size while the flow channel across themembrane is reduced to localize the shear forces at the membraneinterface and reduce the shear forces in the injection ports. Byincreasing the shear forces at the membrane interface, cell migrationcan be improved and cell alteration can be eliminated.

Creating insert supports for both synthetic and natural membranes hasbeen accomplished by using laminates, crimped rings, and adhesives (FIG.19). Laminates and adhesives have primarily been used to support polymermeshes, which in turn are provide mechanical strength to syntheticallyformulated biological membranes. Fabrication using the laminatecomprises sandwiching a stretched mesh between two pieces of polymerlaminates, which are then thermally sealed together. The adhesive methodcomprises stretching a mesh support and adhering a stainless steel ringusing a biocompatible glue. The crimping method, discussed earlier,comprises compressing the membrane between two stainless steel rings.Generally, the laminate and adhesive methods are limited to syntheticmesh-supported membranes, while the crimping method can accommodate bothnatural biological membranes and synthetic meshes.

Example 29 Optically Diagnostic AIS Microfluidic Bioreactor

Immunology has many cascades of events that cannot be observed in anyhuman system at this time. In particular, if a vaccine fails as a resultof a rate-limiting step related to entry into and interactions within animmunological tissue, there is presently no method to measure or improvethis process in humans. To address this problem, an embodiment of thepresent invention include building the AIS in such a way as to be ableto optically monitor in situ the steps of the in vitroimmunological/vaccination process.

In one embodiment, integrated optical waveguides become part of amicro-total analytical system (μTAS) of the AIS, with many differentfunctions including optical excitation, absorption, fluorescence, andimaging on a single microfluidic bioreactor system. An in situdiagnostic system will make optimization and conducting diagnosticevaluations of the immunological constructs more rapid. Two-photonfluorescence can enable visualization of immunological events in allthree dimensions in both artificial and living tissues. This techniquecan aid in understanding and optimizing the effects of variousadjuvants, vaccine candidates, drugs, biologics, biomolecules, andantigen presentation vehicles in vitro and with in situ diagnostics.

Prototype results are presented regarding fabrication of μTAS that canbe used to perform the immunological analysis steps in situ, to simplifythe process and reduce analysis time. In one embodiment, the presentinvention provides an AIS device with the addition of integrated opticalwaveguides for in situ optical diagnostics. These waveguides provideoptical excitation and detection pathways for colorimetric analyses(such as ELISA assays, absorption and fluorescence analysis).

In this example, single layer, planar polymer waveguides were fabricatedusing selective femtosecond laser ablation of a polymer substrate. Aglass slide was coated with an 80 μm-thick layer of a single part,ultraviolet curing polymer with a refractive index of 1.56. After curingfor 30 minutes with a ultraviolet (UV) lamp (4 W), planar opticalwaveguides and microfluidic channels were machined into the polymerusing a Ti:sapphire femtosecond regime laser. The optical waveguides andmicrofluidic channels were each approximately 100 μm wide by 80 μm deep.Light from a CW Nd:YVO4 laser was coupled to the planar waveguidesthrough a 50 μm core diameter optical fiber inserted into a taperedalignment groove as shown on the left. Light guided through the planarwaveguides passes through an intersecting microfluidic channel. Thiswaveguide/channel intersection is shown in the middle with the lasersource off and on the right with the laser source on. Light entering thechannel from the right is collected in the waveguide on the oppositeside of the channel. This light is then coupled to another 50 μm coreoptical fiber and sent to a silicon detector for measurement.

Example 30 In Situ Diagnostic Bioreactor Development

Microfluidic devices that mimic in vivo systems are proving valuable instudying cell interactions and biological processes in vitro. Suchdevices offer several advantages over traditional large-scale fluidicassemblies including small sample and reagent volumes, small wastevolumes, increased surface area-to-volume ratios, low Reynold's numbers(laminar flow), fast sedimentation for particle separation, reducedreaction times, and portability. Some microfluidic devices alsointegrate pumps, valves, filters, mixers, electrodes, and detectors. Theease of alignment and shorter reaction times make near real-timedetection possible using this approach.

Fabrication of microfluidic devices has relied mainly on technologydeveloped in the microelectronics industry, such as photolithography andsubsequent etching of silicon or glass. These technologies often requiremultiple processing steps and clean room facilities and can take days orweeks to produce a working device; they are better suited to massproduction of devices than rapid prototyping. A relatively new method offabrication is ultra-short pulse laser micromachining (USPLM). USPLM hasthe advantage that materials can be machined directly without the needfor masks or photoresist development. Devices can therefore befabricated more quickly, often in a day or less, permitting rapidprototyping. Furthermore, due to the extremely short pulse duration(<150 fs) and high intensities, almost any material can be readilyablated because of multiphoton absorption and ionization, even if it istransparent at the laser wavelength. This is especially useful inmachining materials for an optically transparent bioreactor. FIG. 46shows an ultra-short pulse laser micromachined planar optical waveguidesintegrated into microfluidic channel. Left panel: Tapered port for fiberoptic coupling. Middle panel: microfluidic channel intersection ofplanar waveguide (source off). Right panel: microfluidic channelintersection of planar waveguide (source on, entering from right).

In an embodiment of the present invention, USPLM was used to machinemicrofluidic channels, vias, reservoirs, and integrated opticalwaveguides in the bioreactors. An inexpensive and widely usedbiocompatible silicone elastomer, polydimethylsiloxane (PDMS), comprisesthe main body of the structure. Sheets of PDMS can be patterned by USPLMand then assembled to form the 3D construct (Laser-machined microfluidicbioreactors with printed scaffolds and integrated optical waveguides,Nguyen, et al., Proc. SPIE Int. Soc. Opt. Eng., 5591). The layers may beeither permanently bonded by treating with oxygen plasma or temporarilybonded by applying mechanical pressure. Thus, fabrication of disposableor re-usable devices is easily accomplished

In one embodiment, integrated optical waveguides are fabricated asillustrated in FIG. 39 The waveguides comprise multiple alternatingrefractive index polymer layers in which the middle polymer layer hasthe higher refractive index. In preferred embodiments, the polymers canbe either UV or thermal cured or a combination of both (e.g., PDMScladding and UV curing core). The waveguides are defined by removingmaterial on either side using an ultra-short pulse laser. The laser canalso be used to integrate tapers for fiber optic coupling to thewaveguides. Microfluidic channels are machined either parallel orperpendicular to the waveguides. Light is launched into a waveguide onone side of the microfluidic channel, passed through the channel whereit interacts with the fluid in the channel and then collected by thewaveguide on the opposite side of the channel and sent to a detector. Inanother embodiment, fiber optics are embedded into PDMS and thenmicrofluidic channels machined perpendicular to the fibers, removing asmall section of the fiber in the channel. This eliminates the need forplanar polymer waveguides and fiber-to-waveguide coupling losses at theexpense of elaborate waveguide geometries, such as splitters andcombiners FIG. 40.

FIG. 59 shows an example bioreactor construction with collagen membraneson rings and support matrix. Collagen cushion congealed at 37° C. for 1hour remained highly stable with no collagen degradation for more than 3weeks. Panel A shows the bioreactor design. Panel B shows progressionfrom the whole bioreactor to the level of the collagen matrix cushionwithin the mesh. After the HUVEC cells have reached confluence on thecollagen cushion, the bioreactor is assembled under sterile conditions(Panel C). Once assembled, media flow is initiated.

Example 31 Imaging System

Understanding a complex living system requires a thorough comprehensionof the interactions of cells and their 3D microenvironment.Understanding those cells will necessitate an integrated understandingof all functional units, signal transduction molecules, structuralscaffolding, and genetic material.

Imaging is a powerful unifying tool for such studies. Specifically, inan embodiment of the present invention, confocal microscopy andtwo-photon fluorescence can be integrated in a transparent bioreactorthat houses the vaccination site (VS) and lymphoid tissue equivalent(LTE). Light-microscopic analyses of fluorescently tagged markers mayprovide important information about the location and behavior ofproteins, as well as many details of protein-protein interactions. Atrelatively low resolutions, confocal microscopy can producethree-dimensional (3D) images of fluorescently tagged gene products todetermine their distribution in the cell during different stages of thecell cycle or under various environmental conditions. Such informationallows insights into cell and organelle biology. Furthermore, confocalmicroscopy permits analysis of the ETC 3D architecture, which cannot beachieved by conventional light microscopy. The broad goal is tovisualize cellular constituents and general cytoarchitecture in a stateas close to native organization as possible. As an example, confocalmicroscopy can be used to investigate the DC maturation state in vitrousing integrated, optically transparent bioreactors, where the fiberwaveguides are integrated into the bioreactor.

A need exists to image many biological processes in 3D at thevaccination site construct. For this, 3D confocal imaging of polymersamples can be conducted using two-photon excited fluorescence.Fluorescent molecules may absorb two photons simultaneously beforeemitting light. This phenomenon is referred to as “two-photonexcitation.” Using two-photon excitation in a conventional microscopeprovides several advantages for studying biological samples, includingefficient background rejection, low photo-damage, and depthdiscrimination. A relatively long wavelength of the excitation source(e.g., 798 nm from a mode-locked Ti:Sapphire laser) can be used toenable a larger penetration depth into the 3D ETC than provided byconventional single-photon fluorescence confocal microscopy.

Two-photon confocal microscopy, in conjunction with highly efficientfluorophores, is useful as a tool to study the surface, interface, andinner dimensions of the in vitro vaccination site (VS) and LTE. It canprovide diagnostic information to aid in understanding features such ascell motility towards the VS and within the VS, cell differentiation,and cell maturation to enable optimization of various DC activities.

Several biomarkers are of interest for monitoring in the system. Theseinclude IL-12 (secreted by DCs when they mature: inhibits migration ofDCs), IL-4 (Th2-like responses), CD40, CD40L, CCR7 (migratory chemokinereceptor), IL-1β (inflammatory chemical secreted by immune cells),TNF-α, and VEGF (important modulator of monocyte differentiation intomacrophages or DCs.

Example 32 Complete Design of AIS Device

An example AIS device is illustrated in FIG. 38. The device comprises amicrofluidic bioreactor, ELISA chip with integrated optical waveguides,microfluidic backplane to connect and allow swapping of devices andmicrofluidic connectors for external pumps and reservoirs. Thebioreactor has four external ports, two each above and below the tissueconstruct. An ELISA chip with three sets of two channels is illustrated,though more channels are contemplated in the same footprint in otherembodiments. In each set, one channel is for a sample assay and theother is a control with no sample. Each set is attached to the sameELISA input port, allowing both channels to be prepared simultaneously;however, only one channel in a set is attached to the sample fluid. Thisfluid is pumped from the bioreactor to the ELISA chip through a channelin the microfluidic backplane. Valves control the addition of the samplefluid to each channel. Light is coupled to the ELISA channels throughoptical fibers and the transmitted light is coupled to another fiberattached to a detector. In this preferred embodiment, the bioreactor andELISA chips are both optically transparent for two-photon and confocalmicroscopic examination. In this preferred embodiment, the footprint ofthe entire assembly in this example is approximately 50×75 mm.

Example 33 Magnet Assisted AIS

In a further embodiment the functionality of the AIS is enhanced usingmagnetic microbeads or nanobeads (the magnet assisted AIS, MaAIS).Because the AIS allows monocyte and DC transport between compartments(blood, skin, lymph node) to be accounted for, biomimetic pathways formigration to and from the immunological constructs in the AIS will leadto new insights in vaccine design and better predictive power for theAIS. The two vascular highways are the blood to move monocytes to the VSand lymphatics to move mature DCs from the VS to the LTE.

Guided monocyte and DC migration between compartments viachemoattractants in the flow loops of the bioreactor (with sources atthe VS and LTE, respectively) mimic the natural trafficking propertiesof these cells in vivo, a “biomimetic” solution. In another embodiment,magnetic microbeads and electromagnetic fields can be used as a means todirectly move cells between compartments of the AIS, an “engineering”solution.

Magnetic beads are commonly used as a tool for cell separation, sortingand assay, where the carrier particles bind cells specifically, usuallyvia antigen antibody interactions, or using streptavidin biotincoupling. Magnetic beads typically consist of a magnetite (Fe3O4) orother paramagnetic core of 1-5 μm coated with biocompatible polymers, towhich the affinity groups can be covalently attached. Products of DYNAL(Norway) are, however, macroporous polystyrene particles that aremagnetized by in situ formation of magnetic material inside the pores(Safarik & Safarikova Rev. J. Chromatog. B 722:33-53 (1999); DYNAL(Norway) http://www.dynalbiotech.com/). The micron size of the magneticparticles annuls their ferromagnetism (i.e., the ability to retainmagnetization after removal of the field) so the beads do not cluster.

Localization and separation of these paramagnetic beads is simple andstraightforward; moderate magnetic fields, typically from hundreds tothousands of Gauss, and readily attainable field gradients aresufficient for this (DYNAL (Norway) http://www.dynalbiotech.com/). Manytypes of magnetic beads surface grafted with various antibodies, as wellas with streptavidin, protein A, and other anchoring groups areavailable commercially (Safarik & Safarikova, Rev. J. Chromatog. B722:33-53 (1999); DYNAL (Norway) (http://www.dynalbiotech.com/); AGOWAGMBH (Germany) http://www.agowa.de/struktur/magneticbasis.html). Whenassembled in a confined pool, the beads can be readily transported inaqueous solutions by movement of a pointed magnet. These features allowmagnetic beads to be used to perform various tasks in a microbioreactor, such as selection of cells with appropriate surface markers(receptors; epitopes), transporting selected cells from one area toanother; and bringing cells into contact with a desired environment,including other cells. The use of trafficking magnetic and other fieldcontrolled beads in chemical and biological analyses and syntheses hasbeen recently elaborated in Oestergaard & Bankenstein (1999) WO patentapplication No. WO 99/49319. This approach can be taken a step further.In another embodiment of the present invention, bead trafficking can beimplemented in the design of the AIS.

In this additional embodiment, the magnet assisted AIS includes thefollowing important features:

1. the in vitro device will remain a single unit, preferably flat, withits major elements located in a single isolated volume.

2. most of the available volume will remain filled with, andperiodically recirculated with media containing necessary nutrients,signal molecules and gases.

3. most of the cell trafficking will be directed, with specific beadsused as vehicles (random traveling of cells will be greatly reduced).

4. the beads will be moved by means of pointed permanent orelectromagnets, preferably along the lower surface of the upper plate ofthe flat in the MaAIS construct.

5. major signaling and activation molecules, such as chemokines,maturation signals, antigens can be delivered to the cells by specialbeads in a controlled fashion, if desired.

6. the order of events and the routes of magnetic trafficking can becontrolled by computer.

The MaAIS bioreactor is an extension of the “biomimetic” AIS system. Inan embodiment, the construct is a transparent, flat two plate sandwich.The lower plate harbors specific areas for the VS and LTE sections.These latter elements can be machined as flat depressions in the lowerplate, preferably filled with, e.g., bioconcrete components, regularizedPCL mesh, and matrix (ECM; suitable materials include collagen, fibrinclot, Dermagraft). The plates have special ports for the delivery and/orevacuation of the magnetic beads. The beads can be brought to the portsby computer-controlled deposition heads, similar to those in the BAT.The plates have recirculation channels to provide media flow. How thesystem can be used is schematically illustrated in FIG. 39.

DCs can be attached to the magnetic beads and moved only when thematuration process is complete. This can be achieved using microbeadsfunctionalized with antibodies against mature DC surface markers, (e.g.,CD83, in human cells, which are commercially available).

To allow mature DC-specific labeling of cells exiting the VS afterantigen uptake, magnetic particles functionalized with antibodiesagainst markers upregulated upon DC maturation can be used (including,for example, CD83 (in human cells), CD80, and CD86). In anotherembodiment, pan-DC markers, such as CD11c, can be used to traffic allDCs (mature or not) exiting the VS endothelium into the AIS bioreactorpathway.

Magnetic beads can be also used as nonspecific locomotives, facilitatingcell travel in the fluidic channels of the AIS. An ensemble of magneticbeads sufficiently numerous to partially or substantially fill the crosssection of the fluidic channel can be forced by a computer controlledmagnet to travel around the circular route of the fluidic channel,moving any particulate object along with them. If the beads are coatedwith biocompatible materials and the speed of their movement issufficiently slow (low Re numbers), then they can assist cell movementvia collisions with cells without damage (FIG. 42). In some cases, thiskind of trafficking may be preferable, as no specific receptors on cellswill be involved, minimizing the risk of modulating cell state.

Example 34 Magnetic Bead-Based ELISA for Rapid In Situ Read Out of AISFunction

There are at least two parameters of the immune response to be assessedin the AIS: the titer of specific antibodies produced by activated Bcells, and the total quantity and the ratio of the activated T helpersand cytotoxic T cells (that is, the CD4/CD8 response). In an embodiment,magnetic beads can also be used at this stage, to provide a rapid,computer-controlled, and magnetically actuated assay.

Introduced in the early 1990s (Luk & Lindberg, J. Immunol. Meth. 137:1-8(1991); Gundersen, et al., J. Immunol. Meth. 148, 1-8 (1992)), theso-called Immuno Magnetic Separation ELISA (IMS-ELISA) has drawn recentattention and is now a fast and sensitive immunoassay method (Chou, etal., J. Immunol. Meth. 255:15-22 (2001); Kourilov & Steinitz, Anal.Biochem. 311:166-170 (2002)). For example, Kourilov & Steinitz usedmagnetic beads as solid phase platforms for the attachment of theantibodies to be determined, instead of customary ELISA plates (Anal.Biochem. 311:166-170 (2002)). Secondary antibodies raised against theprimary antibodies and tagged with, for example, alkaline phosphatase orperoxidase were used to titrate the primary targets. This general schemeprovides a mechanism for performing the assay in the AIS device in apotentially fully automated and facile microscale mode.

Example 35 Phagocytosis

Phagocytosis reportedly depends on the size and surface properties ofthe particles in question. In general, microparticles in the size rangeof about 1 to about 3 μm, those that are more hydrophobic; and thosebearing positive surface charges are most likely to be engulfed.Particles bigger than about 5 μm or smaller than about 1 μm, those thatmore hydrophilic, and those bearing negatively charged surfaces are notreadily engulfed by DCs (Chen, et al., J. Colloid Interface Sci. 190:118-133(1998)). Consequently, large and appropriately coated particlescan be used to minimize or effectively stop phagocytosis. The biggestDynabeads particles produced by DYNAL (Norway) are about 5 μm (Safarik &Safarikova, Rev. J. Chromatog. B 722:33-53 (1999); DYNAL (Norway)http://www.dynalbiotech.com/). Much larger beads (10 μm and bigger) areavailable, from, e.g., AGOWA (AGOWA GmbH (Germany)http://www.agowa.de/struktur/magneticbasis.html). Even bigger magneticbeads, structurally resembling the Dynal products, can be synthesizedfrom chitosan (Banchereau & Steinman, Nature 392:245-252, (1998)). FIG.41 illustrates M450 Dynabeads attached to the cells of differing typeand size. Mature DCs are approximately the size of the tumor cellcaptured on the right of the figure, about 30 to 35 μm (Sieben, et al.Comparison of different particles and methods for magnetic isolation ofcirculating tumor cells). As a consequence, it is hard to expect them tobe able to phagocytose beads of, say, about 20 μm size or bigger. FIG.56 show the phagocytosis of microparticles by a monocyte.

Phagocytosis can also be minimized by temporarily decreasing thetemperature of the VS of the AIS to 4° C. This can be achieved, forexample, using a miniature thermoelectric element.

In another embodiment of the present invention, phagocytosis can beallowed to occur, and the cells that have internalized cell specificmagnetic nanoparticles/microparticles can then be moved using a magnet.Magnetic beads made by Dynal (Norway), and by other manufacturers,contain the magnetic material as a minor component of the beads; 80% ormore of the weight, and, accordingly, more than 90% of the volume isoccupied with biocompatible material (Safarik & Safarikova, Rev. J.Chromatog. B 722:33-53 (1999); DYNAL (Norway)(http://www.dynalbiotech.com/); Denkbas, et al., Reactive & FunctionalPolymers 50:225-232 (2002)), which can be made biodegradable withoutloss of other useful properties of the beads. Magnetite (Fe3O4) isbiocompatible.

Example 36 Stimulators of the AIS

The AIS design allows the introduction of peptides or proteins derivedfrom common pathogens or vaccines, including influenza, CMV, or tetanustoxoid. Such antigens can be injected directly into the in vitro VS. Inaddition, different adjuvants or stimulators of the innate immune systemcan be introduced to trigger dendritic cells and other cells to beactivated and induce T and B responses in turn.

Example 37 Measuring Immune Responses in the AIS

After DCs, T and B cells have interacted from about 1 to about 7 days,cells can be extracted from the LTE to assess their properties indetail. In addition, the liquid phase of the LTE and the VS can besampled to measure antibody titers and cytokine/chemokine levels.

(a) T cells: A preferred direct method for gauging antigen specific Tcell activation is tetramer staining Tetramer technology can be used toquantify antigen specific responses if the AIS is populated with cellsof a defined HLA type for which there are available tetramers (e.g.,HLA-A*0201 MP peptide for influenza responses) (Larsson, et al., J.Immunol. 165:1182-1190 (2000); Danke & Kwok, J. Immunol. 171:3163-3169(2003)). T cells can then be stained using the appropriate MHC class Ior II tetramer. In addition, they can also be co-stained intracellularlyfor cytokines (such as IL-2, IFN-γ, IL-4) to assess effector functionsof CD4+ T cells. Additionally, CD8+ T cells can be further tested fortheir ability to lyse target cells pulsed with the same peptide.

For antigens for which associated tetramer staining reagents are notavailable, traditional restimulation approaches can be used to test foran increase in antigen specific T cells, by, for example, looking for[³H]-thymidine incorporation in response to antigen-pulsed, syngeneicdendritic cells. In addition, cytokine production can be measured by,for example, ELISPOT and intracellular staining, and CTL lysis bystandard target lysis methods. A more general method that can be used tostudy T cell proliferation is staining input T cells with CFSE dye,which allows quantification of cell division by measuring CFSE dilutionusing flow cytometry (Hasbold, et al., Immunol. Cell. Biol. 77:516-522(1999)).

(b) B cells: In parallel, B cell responses and antibody titers in the‘serum’ or fluid of the artificial immune system can be measured

(c) Dendritic cells: Dendritic cells can be isolated from about severalhours to about several days after immunization and tested for, forexample, viability, maturation marker expression, and functionality. Itis anticipated that DCs will change their properties, depending on theinitial vaccine stimulus.

(d) Cytokines and chemokines. It will also be important to assess thelevels of key cytokines (including IFN-γ, IL-12, IFN-α, IL-2,) andchemokines (including ELC, BLC) that are present during the cascade ofvaccine action, to gauge the efficacy of a vaccine.

Example 38 Titration of Vaccine-Specific Antibodies

B cells cultivated in the LTE are expected to produce antibodies (Abs)in response to the immunization of the AIS device with a tested vaccine.Magnetic beads tagged with the antigen of immunization (e.g., ovalbumin)should bind to these antibodies. Secondary antibodies raised against theprimary Abs and tagged with enzymatic or fluorescent reporting groupscan be used to form a traditional ELISA sandwich, allowing determinationof the level of the primary Abs (FIG. 43).

Example 39 Titration of the CD4/CD8 T Cells

Appearance of the CD4+ and CD8+ markers on T cells is an outcome measureof their DC-induced activation. To employ an IMS-ELISA sandwich schemefor a CD4/CD8 assay, it is important to anchor the activated T cells tomagnetic beads; this can be achieved using CD3 or CD2 activationmarkers. It is known, however, that binding antibodies to these markersper se typically initiates activation of T cells, which is undesirablein the assessment of T cell activity. On the other hand, the time periodnecessary to activate T cells via Ab attachment to CD3 is about 2 to 4days (Protocol for anti-CD3 activation of T cells from E-Bioscience (SanDiego, Calif.)(http://www.ebioscience.com/ebioscience/appls/AC145.htm#human)).Consequently, an assay performed in a significantly shorter time willlikely still be informative. A magnetic bead/T cell conjugate formed viaattachment of the bead to CD2 or CD3 will be a target for anti-CD4 andanti-CD8 antibodies tagged with specific labels, preferably withfluorescent groups (FIG. 44).

In general, more sophisticated and less routine multi target assessmentsof T and B cell activity can be performed for the AIS device with theaid the magnetic beads and sandwich ELISA techniques.

Example 40 Rapid Vaccine Assessment: How the AIS can be Used to AssessVaccines

A more accurate in vitro model for the assessment of human vaccines for,e.g., bio-warfare agents, emerging infectious diseases, and currentglobal epidemics, is presented. The following example illustrates howthe AIS can be used to assess efficacy and mechanisms of vaccines andother immunotherapies.

Traditional vaccine formulation starts with attenuation of a viral orbacterial strain, to reduce infectivity and pathogenic effects, whilepreserving immunogenicity and adjuvanticity Important features of avaccine are thus to provide target antigenic epitopes for neutralizingantibodies and for CTL responses and adjuvant activity to stimulate apotent B and T cell memory response. However, there is no formula forreliably designing optimal epitopes or adjuvants; it remains largelyempirical. The AIS can aid in discovering the rate-determining steps invaccine efficacy and providing data to enable improved vaccine designand formulations.

A use of the AIS is in rapid screening of vaccine formulations to findoptimal methods for antigen delivery and adjuvant stimulation of a humanimmune response. In particular, with the AIS, the efficacy of a vaccinecan be tested in a more physiological context, thus providing animprovement over the predictive power of current testing methodologies.The activity of vaccines at each step of the vaccine life cycle can bemeasured, thus helping to determine which steps are important forvaccine success and failure.

Using the AIS, it is possible to quantitatively assess T and B cellstimulation in the context of more physiological environment than thatfound in a tissue culture dish or a non-human animal. Specifically:

1. by providing a venue in the LTE for DCs, CD4+ T , CD8+ T and B cellsto meet, it can be determined whether a candidate vaccine promotesoptimal levels of T cell help to induce CTL and B cell responses;

2. by allowing DCs, T and B cells to meet in a 3D environment withextracellular matrix and support cells, the LTE more realisticallymimics the environment of the lymph node where the triad of cellsinteract in vivo;

3. the inclusion of endothelium ensures that monocytes and DCs interactwith endothelial cells during recruitment and emigration; theseinteractions require the expression of specific proteins on the surfaceof immune and endothelial cells, some of which may be sensitive to thevaccine candidate and thus affect vaccine efficacy in humans; and

4. the presence of a more representative population of cells and ofcells that must migrate across the endothelium and differentiate inresponse to local tissue signals, will lead to more accurate results(for example, it will be possible to distinguish the effects of TLR9(Tol-like receptor 9) ligands versus TLR4 ligands as they are expresseddifferentially on multiple DC subtypes that may not be present in 2Dcultures and that are known to be different in their Tlr expression inmice (Kadowaki, et al., J. Exp. Med. 194:863-869 (2001)).

The AIS of the present invention allows more accurate readouts becauseit contains a representative distribution of different cell types,opportunities for typical cell-cell interactions, basal activationstates of cells that mimic cells in living tissues, and a more natural3D extracellular matrix to support cell behavior and function.

The AIS enables the measurement of many important early and acuteparameters of the response in the VS, and later parameters of theresponse in the LTE. Such measurements would be almost impossible tomake in human clinical trials. The ability to measure many parameterswill allow identification of steps that differ between vaccinecandidates and will enable rational change and optimization of thevaccine candidate.

Measurements that can be made with the AIS include:

1. monocyte recruitment;

2. differentiation of DC subtypes;

3. DC antigen loading;

4. DC maturation;

5. DC emigration;

6. endothelium activation and function;

7. kinetics and numbers of DCs arriving into lymph node;

8. efficiency of interactions between T cells and DCs;

9. efficiency of interactions between B cells and DCs;

10. efficiency of interactions between T and B cells and DCs; and

11. activation status of T and B cells.

Differences in efficacy among vaccine candidates may be due to theirdifferential ability to modulate any one of these or other steps.Identification of the steps that differ in successful and failedvaccines will allow a more rational model of how vaccines work.

For example, FIG. 45 shows the role of CCR8 in monocyte immigration inboth in vitro and in vivo models. In panels A and C, monocytes wereco-cultured with endothelial cells grown on a type I collagen gel for 48h, permitting the separation of the population intoreverse-transmigrating DCs or macrophages that remain in thesubendothelium. Inclusion of neutralizing anti-CCR8 mAb 3B10 during theassay period when monocytes traverse endothelium in the apical-to-basaldirection had no effect (panel A), but 3B10 anti-CCR8 mAb and anti-CCR8mAb 5B11 significantly inhibited reverse transmigration in more than 5independent experiments (panel C). In panels B and D, green fluorescentlatex microspheres were injected into the skin of CCR8-deficient micethat were compared to age- and sex-matched wild-type C57BL/6counterparts. This method traces monocyte conversion into DCs thatpresent antigen in draining lymph nodes. Ungated day 1 skin analysisshows the cell suspension recovered from the site of injection.Fluorescent latex is found in a population of infiltrating monocytes inboth wild-type and CCR8 knock-out mice. The number of DCs bearing 2 ormore latex particles in the draining lymph nodes was quantified 3 dayslater. To combine data from different experiments, the mean number ofmigrated cells in wild type mice was set equal to 1.0 for eachexperiment and relative values for all wild type and knock-outindividuals in that experiment were calculated. Lymph node dot plotsshow MHC II (I-Ab) and Gr-1 levels in lymph nodes and skin of wild typeand CCR8−/− mice. These are quantitative comparisons, as they depict theentire population of latex-bearing cells recovered from pooled brachiallymph nodes from individual mice.

Example 41 Utilizing AIS as a Biofactory

In an embodiment of the present invention, the assembled LTE is used asa “biofactory,” biosynthesizing various desired biomolecules (such ascytokines, proteins, antibodies). For example, if an antigen ispresented to B cells, they can create antibodies in the LTE.Potentially, the created antibodies could also be monoclonal, dependingon the repertoire of B cells and how the peptide is presented to the Bcells. Monoclonal antibodies (mAb) are used extensively in basicbiomedical research, in diagnosis of disease, and in treatment ofillnesses, such as infections and cancer. Antibodies are important toolsused by many investigators in their research and have led to manymedical advances.

Example 42 T Cell Motility Induction in Inverse Opal Scaffolds

Among the multiple factors influencing naive T cell migration, adhesionmolecules and chemokines play a significant role, and inclusion of thecorrect balance of these factors in an inverse opal hydrogel LTE allowsnaive T cell motility, quantitatively similar to in vivo cell migration,to be stimulated. As an example, fluorescently-labeled, mature murinedendritic cells (generated from the bone marrow of C57BL/6 mice by themethod of Inaba, et al., J. Exp. Med., 176:1693 (1992) and matured bytreatment with LPS for 12 hrs) and labeled naive CD4+ T cells from thelymph nodes of OT-II transgenic mice were added to inverse opalscaffolds coated with fibronectin. It is postulated that chemokinesproduced by mature dendritic cells (e.g., CCL19), and adhesion moleculesexpressed on the surface of these cells would promote naive T cellmotility. Time-lapse 3D videomicroscopy was used to record the dynamicsof cells within the scaffold over 2 hrs. T cells were highly active andexhibited ‘start-stop’ migration patterns reminiscent of migrationbehavior observed by intravital imaging studies of T cell migration inmouse lymph nodes. As shown in FIG. 61, T cells could migrate from voidto void over the surface of dendritic cells in the scaffold, bothvertically (FIG. 61A), and laterally (FIG. 61B). Quantitation of thecell paths and velocities (FIG. 62) showed that the cells moved with anaverage velocity of 4.6 μm/min, approaching the values reported in vivofor naive T cells, and that T cells had maximal ‘bursts’ of speed up to30 μm/min, as seen in vivo. Naive T cells alone at lymph-node-likedensities in inverse opal scaffolds failed to polarize or migrate (FIG.63), indicating the need for a more complete lymphoid microenvironmentto stimulate cell migration.

Example 43 Sorption Capacity of Heparin-Saturated Cytopore

Cytopore aliquots (0.4 ml, tightly sedimented) were treated with heparinand thoroughly washed and incubated in different concentrations of BLCin 3 ml PBS/BSA and washed. Afterwards, BLC remaining in solution andBLC absorbed by Cytopore/heparin were determined separately. As shown inFIG. 67, the sorption curve of Cytopore/heparin appeared sufficientlylinear. Consequently, the conditions used were likely far fromsaturating the Cytopore with the BLC chemokine. This suggests Cytoporesaturated with heparin as described, can potentially carry much higher(˜10 times or more) loads of chemokines than in the example experiments.

Example 44 Kinetic in Silico Modeling of T and B Interaction in LTE

As shown in FIG. 57, B and T cells were modeled in 2D space as physicalobjects endowed with capability to walk randomly and come in physicalcontact with each other for a certain time. It was found that at areasonably high concentration, for example at ˜3×10⁷ cells/ml, the LTEmodel containing microcarriers as centers of self-concentration of Bcells and T cells randomly walking around has an advantage towards themodel of randomly distributed and co-cultured B and T cells: T cells hadsufficiently higher probability to come in contact with B cells in theLTE model. This advantage gradually decreased with further increasingthe cell concentration, and became negligible at concentration ˜3×10⁸cells/ml, which correspond to dense slurry of cells (FIG. 57). Thiscomputational result is considered circumstantial evidence in favor ofLTE designs containing areas of concentrated B cells (mimicking thegerminal centers of the natural lymph node) and loosely migrating Tcells.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it that will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1. A method of producing a biomolecule using an in vitro two culturecellular system, comprising: a) adding an exogenous antigen of interestto a vaccination site (VS) culture, wherein the VS culture comprises afirst substantially planar matrix, a plurality of cells consisting ofendothelial cells and/or epithelial cells attached to said first matrixand a population of peripheral blood mononuclear cells (PBMCs); b)culturing the VS culture of a) in the presence of the antigen for aduration of time sufficient to mature dendritic cell precursors presentin the population of PBMCs; c) transferring dendritic cells matured inthe VS culture of b) to a three-dimensional artificial lymphoid tissueequivalent (LTE) culture, wherein the LTE culture comprises a secondmatrix and a plurality of lymphocytes attached to said second matrix; d)culturing the LTE culture of c) for a duration of time sufficient forlymphocyte activation; and e) harvesting a biomolecule produced by theactivated lymphocytes, wherein said biomolecule is selected from thegroup consisting of cytokines, proteins and antibodies.
 2. The method ofclaim 1, wherein said biomolecule is an antibody.
 3. The method of claim1, wherein said plurality of cells forms a vascular endothelium on oneside of said first matrix.
 4. The method of claim 1, wherein saidplurality of cells forms a vascular endothelium on both sides of saidfirst matrix.
 5. The method of claim 1, wherein said plurality of cellsforms a vascular endothelium on one side of said first matrix and anepithelium on an opposing side of said first matrix.
 6. The method ofclaim 1, wherein said plurality of cells forms a vascular endothelium onone side of said first matrix and a lymphatic endothelium on an opposingside of said first matrix.
 7. The method of claim 1, wherein said firstmatrix comprises a natural biopolymer.
 8. The method of claim 7, whereinsaid biopolymer is selected from the group consisting of type I rat tailcollagen, bovine type I collagen, and chitosan.
 9. The method of claim1, wherein said first matrix comprises bovine type I collagen matrixdeposited and congealed on a nylon mesh.
 10. The method of claim 1,wherein said first matrix is selected from the group consisting of axenographic extracellular matrix (ECM) sheet, natively polymerized humanamniotic connective tissue, reconstituted collagen matrix, andchitosan/collagen membrane scaffolds.
 11. The method of claim 1, whereinsaid plurality of cells attached to said first matrix comprises humancells.
 12. The method of claim 11, wherein said human cells comprisehuman vascular endothelial cells (HUVECs).
 13. The method of claim 1,wherein said plurality of cells attached to said first matrix consistsof endothelial and/or epithelial cells derived from embryonic stemcells.
 14. The method of claim 1, wherein said endothelial cells arehuman skin-derived vascular endothelial cells or said endothelial cellsare vascular and lymphatic endothelial cells.
 15. The method of claim 1,wherein said plurality of lymphocytes comprises T cells and B cells. 16.The method of claim 15, wherein said plurality of lymphocytes furthercomprises dendritic cells.
 17. The method of claim 1, wherein saidplurality of lymphocytes comprises naive T cells and naive B cells. 18.The method of claim 1, wherein said plurality of lymphocytes comprisesmemory T cells and memory B cells.
 19. The method of claim 1, whereinsaid plurality of lymphocytes further comprises lymphoid stromal cells.20. The method of claim 19, wherein said lymphoid stromal cells arederived from lymph node fragments, lymph node, spleen, or tonsil. 21.The method of claim 1, wherein said plurality of lymphocytes comprises Bcells and T cells negatively selected from peripheral blood lymphocytes.22. The method of claim 1, wherein said plurality of lymphocytesattached to said second matrix comprises cells derived from embryonicstem cells.
 23. The method of claim 1, wherein said second matrixcomprises natural or synthetic ECM materials.
 24. The method of claim 1,wherein said second matrix comprises natural or synthetic lymphoidECM-derived hydrogel.
 25. The method of claim 1, wherein said secondmatrix comprises synthetic ordered macroporous hydrogel, wherein saidhydrogel comprises poly(ethylene glycol) (PEG) dimethacrylate, PEGpeptide PEG block copolymers and ordered colloidal crystal of poly(methyl methacrylate) latex microspheres.
 26. The method of claim 1,wherein said second matrix comprises segregated T cell and B cell zones.27. The method of claim 26, wherein said T cell zone comprises naive Tcells and collagen fibers.
 28. The method of claim 27, wherein saidcollagen fibers comprise collagen I, collagen III or fibronectin. 29.The method of claim 26, wherein said segregated T cell and B cell zonesare fabricated by combined action of digital printing and controlledrelease of chemoattractants.
 30. The method of claim 26, wherein saidsegregated T cell and B cell zones are fabricated by digital printing orsaid segregated T cell and B cell zones are fabricated by controlledrelease of chemoattractants.